CN115948417A - Barley HvFRF1 gene, protein, expression vector and application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a barley HvFRF1 gene, a protein, an expression vector and application. The nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1. According to the protein coded by the barley HvFRF1 gene, the amino acid sequence of the protein is shown as SEQ ID NO. 2. The invention also provides application of the barley HvFRF1 gene in screening arabidopsis thaliana varieties and barley varieties by regulating and controlling the drought resistance of the arabidopsis thaliana and the barley. The invention verifies the function of the gene on arabidopsis thaliana and barley by cloning and analyzing the barley HvFRF1 gene and combining with arabidopsis thaliana and barley transgenic technology. The over-expression of the HvFRF1 gene is found to be capable of obviously improving the tolerance of arabidopsis thaliana and barley plants to drought stress. The invention provides theoretical basis and related genes for drought-resistant breeding and production of barley.

Description

A kind of barley HvFRF1 gene, protein, expression vector and application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a barley HvFRF1 gene, protein, expression vector and application.

Background technique

Drought is the most important climate disaster affecting human production and crop growth. It occurs frequently, lasts long, and affects a wide range (Mosley et al., 2015; Caine et al., 2019). In recent years, with global warming and extreme climate change abnormalities, drought has become increasingly severe and has become the most important limiting factor of food production in the world, seriously threatening food security. According to the Emergency Disaster Database (EM-DAT, https://www.emdat.be/), the frequency of droughts accounts for one-twentieth of the global natural disasters, and the direct losses of all natural disasters account for more than a third. Therefore, crop drought research has received special attention from all over the world, and cultivating drought-resistant crop varieties to improve crop drought resistance has very important practical significance for making full use of land and natural resources and promoting sustainable agricultural development.

Barley (Hordeum vulgare L.) is one of the oldest diploid crops in the world and is suitable for cultivation in high altitude, cold, drought, saline-alkali, and barren land (Carter et al., 2019; Kebede et al., 2019). Competing with staple crops for arable land resources is of great significance in coping with global climate change and ensuring regional food security. In the past five years, the average annual planting area of barley in my country was basically stable at 900,000 to 1,000,000 hectares, with a total output of 4.5 to 5 million tons, while the annual consumption of barley was nearly 15 million tons, and the largest annual import volume exceeded 10 million tons (FAOSTAT, http: http://faostat.fao.org/). Barley has a wide range of uses, including food, feed, brewing, medicinal and other functions. Because barley is rich in nutrition and has health care effects, it has become an ideal raw material for processing popular, economical, convenient and therapeutic foods. Therefore, mining drought-resistant germplasm and key drought-resistant genes of barley and clarifying its drought-resistant mechanism is of great significance for developing new ways to resist drought stress and improving crop drought resistance and yield.

FRS is a new class of transcription factors, produced by "molecular domestication" of one or more loss-of-function MULE transposons (Lin et al., 2007), whose family members are regulated by transcriptionally activating or repressing the expression of different target genes Various cellular processes play important functions in plant growth and development and stress resistance (Ouyang et al., 2011; Li et al., 2016; Ritter et al., 2017).

Through the above analysis, the problems and defects in the prior art are:

FRF is a kind of truncated FRS, which competes with FRS for DNA binding sites, thereby regulating the expression of target genes. However, the research on this kind of genes is mainly concentrated on Arabidopsis, and there are few reports on the FRF genes of crops. It cannot provide a theoretical basis for drought-resistant breeding and production of barley; therefore, identifying barley FRF genes and exploring their functions in regulating drought stress are of great significance for improving crop resistance to drought stress and genetic improvement of crops.

Contents of the invention

In order to overcome the problems existing in the related technologies, the disclosed embodiments of the present invention provide a barley HvFRF1 gene, protein, expression vector and application. It specifically relates to a barley HvFRF1 gene and its application.

The technical scheme is as follows: a barley HvFRF1 gene, the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO.1.

In one embodiment, the barley HvFRF1 gene is a gene cloned from barley.

Another object of the present invention is to provide a protein encoded by the barley HvFRF1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

Another object of the present invention is to provide an Arabidopsis overexpression vector super1300:HvFRF1 constructed by homologous recombination based on the barley HvFRF1 gene. The nucleotide sequence is shown in SEQ ID NO.26.

Another object of the present invention is to provide a barley overexpression vector pBract214:HvFRF1 constructed by the Gateway method based on the barley HvFRF1 gene. The nucleotide sequence is shown in SEQ ID NO.29.

Another object of the present invention is to provide an application of barley HvFRF1 gene in obtaining drought-resistant Arabidopsis strains.

Another object of the present invention is to provide an application of the barley HvFRF1 gene in obtaining drought-resistant barley lines.

Another object of the present invention is to provide an application of barley HvFRF1 gene in tissue expression in barley root system induced by drought stress.

Another object of the present invention is to provide a use of barley HvFRF1 gene in regulating Arabidopsis thaliana and barley drought resistance screening Arabidopsis varieties and barley varieties.

In combination with all the above-mentioned technical solutions, the advantages and positive effects of the present invention are:

First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, closely combine the technical solution to be protected in the present invention and the results and data in the research and development process, etc., to analyze in detail and profoundly how to solve the technical solution of the present invention Technical problems, some creative technical effects brought about after solving the problems. The specific description is as follows:

With the frequent and intensified drought caused by global warming, drought has become the most important limiting factor of food production in the world and seriously threatens food security. The research on crop drought resistance has attracted extensive attention from all over the world. Mining genes related to crop drought resistance and cultivating drought-resistant crop varieties to meet the demand for drought-resistant crops due to global climate change and population growth are of great significance for sustainable agricultural development and food security. However, due to the genetic complexity of drought resistance traits, it has become an important bottleneck for analyzing the genetic basis of crop drought resistance and cultivating breakthrough drought-resistant (tolerant) crop varieties. Barley is the fourth largest cereal crop in the world. It is suitable for planting in drought, saline-alkali, barren and other lands. It is an important material for excavating excellent drought-resistant genes. The present invention clones and analyzes barley HvFRF1 gene, and combines Arabidopsis thaliana and barley transgenic overexpression technology to verify the function of the gene. The results show that overexpression of HvFRF1 gene can significantly improve the drought resistance of Arabidopsis thaliana and barley. The invention can provide excellent genetic resources for drought-resistant genetic improvement of barley and other crops, innovate and enrich crop drought-resistant genetic regulation theory, and has important guiding significance for breeding excellent new varieties of drought-resistant crops.

Second, regarding the technical solution as a whole or from the perspective of a product, the technical effects and advantages of the technical solution to be protected by the present invention are specifically described as follows:

The invention discloses the barley HvFRF1 gene and its application in regulating the tolerance of Arabidopsis and barley to drought stress. The nucleotide sequence of the CDS region of the barley HvFRF1 gene is shown in SEQ ID No.1. The present invention Through the cloning and analysis of the barley HvFRF1 gene, combined with Arabidopsis and barley transgenic technology, the function of the gene was verified in Arabidopsis and barley. It was found that overexpression of HvFRF1 gene can significantly improve the tolerance of Arabidopsis and barley plants to drought stress. The invention provides a theoretical basis and related genes for drought-resistant breeding and production of barley.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the disclosure and together with the description serve to explain the principles of the disclosure.

Fig. 1 is a flowchart of a method for constructing a super1300:HvFRF1 vector provided by the embodiment of the invention provided by the embodiment of the present invention;

Fig. 2A is the prediction result of HvFRF1 functional domain in the HvFRF1 functional domain and conserved sequence analysis provided by the embodiment of the present invention, including a typical FAR1 functional domain diagram;

Fig. 2B is the results of protein sequence alignment between barley HvFRF1 and Arabidopsis AtFRF1, AtFRF2, AtFRF3, and AtFRF4 in the HvFRF1 functional domain and conserved sequence analysis provided by the embodiment of the present invention. The box marks the location of the FAR1 functional domain, and the triangle marks the Conserved amino acids in the FAR1 functional domain;

Fig. 3A is the phylogenetic tree analysis diagram of HvFRF1 and homologous proteins in Arabidopsis thaliana, rice and wheat in the evolution analysis of HvFRF1 gene provided in the embodiment of the present invention and the expression analysis in response to drought stress. FRF members are closely related;

Figure 3B is a graph of the expression of the HvFRF1 gene after 7 days and 14 days of drought stress in the evolution analysis of the HvFRF1 gene provided in the examples of the present invention and the expression analysis in response to drought stress. With the extension of the drought stress time, the HvFRF1 gene is induced to up-regulate the expression fold bigger;

Figure 4A is a graph showing the expression of the HvHRF1 gene in the barley seedling and tillering stages analyzed by qRT-PCR in the expression analysis of the HvHRF1 gene provided in the embodiment of the present invention, and the expression level of the gene in the barley root is higher;

Figure 4B is a map of the localization of the HvFRF1 gene at the subcellular level of Arabidopsis protoplasts in the expression analysis of the HvHRF1 gene provided by the embodiment of the present invention, the gene is mainly localized in the nucleus, and the red fluorescence is the nucleus localization marker; Bar=10 μm;

Figure 4C is a map of the expression sites of the HvHRF1 gene in barley roots and leaves in the in situ PCR analysis of the expression analysis of the HvHRF1 gene provided by the embodiment of the present invention, and the gene is mainly highly expressed in the epidermal cells of the root and the vascular bundles of the root and the leaf; e , epidermis, epidermis; c, cortex, cortical parenchyma; en, endodermis, endodermis; x, xylem, xylem; xp, xylem parenchyma, xylem parenchyma; p, phloem, phloem; bs, Bundle sheath; vascular bundle sheath; xv, xylem vessel, xylem vessel; pv, phloem vessel, phloem vessel; ms, mesophyll, mesophyll; Bar=50 μm;

Figure 5A is a schematic diagram of the construction of the Arabidopsis expression vector provided by the embodiment of the present invention and the Arabidopsis overexpression vector super1300:HvFRF1 in the seedling phenotype of the HvFRF1 transgenic Arabidopsis line and WT under normal watering and drought treatment conditions Schematic diagram of the construction; the super promoter drives the HvFRF1 gene, the 35S promoter drives the resistance gene Hygromycin, and NOS represents the terminator;

Figure 5B is a schematic diagram of the construction of the Arabidopsis expression vector provided by the embodiment of the present invention and the overexpression of Arabidopsis lines OE1 and OE2 in the seedling phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought conditions , drought resistance phenotypes of OE3 and WT after drought treatment;

Figure 5C is a schematic diagram of the construction of the Arabidopsis expression vector provided by the example of the present invention and the overexpression of Arabidopsis lines OE1 and OE2 in the seedling phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought conditions , MDA content map of OE3 and WT after drought treatment;

Figure 5D is a schematic diagram of the construction of the Arabidopsis expression vector provided by the embodiment of the present invention and the overexpression of the Arabidopsis lines OE1 and OE2 in the seedling phenotypes of HvFRF1 transgenic Arabidopsis lines and WT under normal watering and drought conditions , Proline content map of OE3 and WT after drought treatment;

Figure 6A is a schematic diagram of the construction of the barley overexpression vector provided by the embodiment of the present invention and a schematic diagram of the construction of the barley overexpression vector pBract214:HvFRF1 in the seedling phenotypes of transgenic barley lines and WT under normal watering and drought treatment; 35S promoter drive The resistance gene Hygromycin, the maize Ubi promoter (ZmUbi) drives the HvFRF1 gene, attR1 and attR2 are the LR reaction exchange positions in the Gateway method, and NOS represents the terminator;

Figure 6B is a schematic diagram of the construction of the barley overexpression vector provided by the embodiment of the present invention and the seedling phenotypes of the transgenic barley lines and WT under normal watering and drought treatment. Among the overexpressed barley lines OE1, OE2 and WT after drought treatment drought resistance phenotype;

Figure 6C is a schematic diagram of the construction of the barley overexpression vector provided by the embodiment of the present invention and the expression of the HvFRF1 gene in the overexpressed barley lines OE1, OE2 and WT in the seedling phenotypes of the transgenic barley lines and WT under normal watering and drought treatment quantity;

Figure 6D is a schematic diagram of the construction of the barley overexpression vector provided by the embodiment of the present invention and the seedling phenotypes of the transgenic barley lines and WT under normal watering and drought treatment. Among the overexpressed barley lines OE1, OE2 and WT after drought treatment The dry weight of the aboveground part.

Detailed ways

In order to make the above objects, features and advantages of the present invention more comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

1. Example of explanation:

The embodiment of the present invention provides a drought-resistant gene cloned from barley, which provides related genes and theoretical basis for breeding drought-resistant barley varieties.

The embodiment of the present invention also provides a kind of application of barley HvFRF1 gene in regulating Arabidopsis thaliana and barley drought resistance to screen Arabidopsis varieties and barley varieties, the nucleotide sequence of the CDS region of the barley HvFRF1 gene is as SEQ ID NO. 1, the amino acid sequence encoded by the HvFRF1 gene is shown in SEQ ID NO.2.

The related sequences include:

CDS nucleotide sequence SEQ ID NO.1: ATGCCGGAGGTGATGTCCACGCCACAGCAACCCACAGCCATGATGAGATTTGACACTCTTGAGGATGCTGAGAAACACTACAAACTGTATGCGAGGCAGAAAGGTTTTGGAGTAAGGTATTGTTTCCGAAAAAAGGTCAGAGGCTAGTGGTGAACTGATAAGAGCATCACTTGTCTGCCATAGAGCTGGGTTGA AGATCAAGAGGAAAGTAGACACCCAAAACCCGCAACCTATTGCCCCTGAGAGGAGTAGGAATACAACTGAAAGAACAAACTGCCCAGCCCGTATGTTTGTGAAGCGAAGAGATAATGCCTGGGTTGTAACAGAAATAAATGACAACCCATAACCACCTCTCATAAAGAAATGGTCGCTGACAGGATACCTACGATCACATAGACATATCCCTGAAGAAGAACAACAGTTT GTGAAGTTGCTTCATTCGTGCAATCTAGAACCTTCTAGACAGATGCAGTTGTTGACAGAGTTGCATGGCCAACGTGAAGCCATTGGTTACACTGACAAAGACTTGGCAAATTTGCTAGCAAAGTTCCGGGCTGAGCACAAATACACTGATATGCAGGACACGATTGAGTACTTCAAAAAGCAGTCAACAGCTTGATAAAGACTTTTTTTACAAGTACAAGCTTGATGAT GAAAATAAGGTCCAATGCATTTACTGGATTGATGGTTCAGCGAGAAGGGCTTACAAGTTTTTCAGTGATTGTGTCTCTTTTGACACAACATACTTGACCAATATATACAAGATGCCTTGTGCCCGTTTATTGGTATAA.

Amino acid sequence SEQ ID NO.2: MPEVMSTPQQPTAMMRFDTLEDAEKHYKLYARQKGFGVRYCFRKRSEASGELIRASLVCHRAGLKIKRKVDTQNPQPIAPERSRNTTERTNCPARMFVKRRDNAWVVTEINDNHNHPLIKKWSLTGYLRSHRHIPEEEQQFVKLLHSCNLEPSRQMQLLTELHGQRE AIGYTDKDLANLLAKFRAEHKYTDMQDTIEYFKSSQQLDKDFFYKYKLDDENKVQCIYWIDGSARRAYKFFSDCVSFDTTYLTNIYKMPCARLLV.

The embodiment of the present invention also provides an overexpression vector super1300:HvFRF1 and pBract214:HvFRF1.

Among them, super1300 is an Arabidopsis overexpression vector preserved in our laboratory, and pBract214 is a barley overexpression vector provided by Dr. Peter Dominy of the University of Glasgow, UK. The present invention connects the HvFRF1 gene into the above two vectors through homologous recombination and Gateway methods respectively, and successfully constructs the Arabidopsis thaliana overexpression vector super1300:HvFRF1 (SEQ ID NO.26) and the barley overexpression vector pBract214 of the HvFRF1 gene: HvFRF1 (SEQ ID NO.29), can verify the function of HvFRF1 gene in Arabidopsis and barley respectively.

As shown in Figure 1, the embodiment of the present invention provides a method for constructing a super1300:HvFRF1 vector, comprising the following steps:

S101, using the preserved pEASY:HvFRF1 plasmid as a template, design a pair of primers HvFRF1-OE-F1 and HvFRF1-OE-R1 to amplify the ORF fragment of the HvFRF1 gene; the primer sequences are: SEQ ID NO.15, SEQ ID NO.16 ;

HD Cloning Kit同源重组试剂盒(Takara，日本)将胶回收纯化的ORF片段与superl300载体(本实验室保存)进行同源重组反应；将反应产物转化大肠杆菌DH5α，阳性克隆送公司测序，测序正确的单克隆提质粒保存，所得质粒为superl300:HvFRF1；S102, using

HD Cloning Kit homologous recombination kit (Takara, Japan) carried out homologous recombination reaction between the ORF fragment recovered and purified from the gel and the superl300 vector (preserved in our laboratory); the reaction product was transformed into Escherichia coli DH5α, and the positive clone was sent to the company for sequencing. The correct monoclonal extraction plasmid is preserved, and the resulting plasmid is superl300:HvFRF1;

S103, transform the above plasmids into Agrobacterium competent cells EHA105, spread rifampicin+kanamycin-resistant YEB plates, culture at 28°C for about 40-48 hours, and verify positive clones by PCR; add an equal volume of positive clone bacteria solution 30% sterilized glycerol, mixed evenly, quickly frozen in liquid nitrogen, and stored at -80°C for later use.

The embodiment of the present invention also provides a pBract214:HvFRF1 vector construction method comprising the following steps:

The Gateway method was used to construct the overexpression vector of HvFRF1 gene, namely pBract214:HvFRF1. Using the saved pEASY:HvFRF1 plasmid as a template, a pair of primers HvFRF1-OE-F2 and HvFRF1-OE-R2 were designed to amplify the ORF fragment of HvFRF1 gene. The primer sequences are: SEQ ID NO.21, SEQ ID NO.22;

The PCR amplification products were detected by 1% agarose gel electrophoresis, the target products were recovered and purified by gel, and the concentration of the recovered products was measured. Subsequently, Gateway BP Clonase II enzyme mix (Invitrogen, USA) was used to perform BP recombination reaction with the gene recovery product of known concentration and the pDONR (Zeo) entry vector.

React at 25°C for 4 hours, add 1 μL of Proteinase K, continue to react at 37°C for 10 minutes, the reaction product is immediately transformed into E. coli DH5α competent cells, coated with bleomycin-resistant LB solid medium, cultured at 37°C for about 16 hours, picked Monoclonal, shake the bacteria for bacterial fluid PCR, send the bacterial fluid of positive clones to the company for sequencing, the sequenced correct single clone is multiplied, the bacterial fluid is stored in glycerol and the extracted plasmid is preserved, and the plasmid is named pDONR(Zeo):HvFRF1.

Gateway LR Clonase II enzyme mix (Invitrogen, USA) was used to carry out LR recombination reaction between the above-mentioned known concentration of the plasmid and the target vector pBract214 (provided by Dr. Peter Dominy, University of Glasgow, UK).

React at 25°C for 4 hours, add 1 μl of Proteinase K, continue to react at 37°C for 10 minutes, the reaction product is immediately transformed into E. coli DH5α competent cells, coated with kanamycin-resistant LB solid medium, cultured at 37°C for about 16 hours, picked Monoclonal, shake the bacteria for bacterial liquid PCR, send the bacterial liquid with the target band to the company for sequencing, the sequenced correct single clone is multiplied, the bacterial liquid is stored in glycerol and the extracted plasmid is preserved, and the plasmids are respectively named pBract214:HvFRF1;

The correctly sequenced pBract214:HvFRF1 plasmid and pSoup plasmid were co-transformed into Agrobacterium competent cell AGL1, coated with rifampicin+kanamycin-resistant YEB plate, cultured at 28°C for about 40-48h, and PCR verified positive clones. Take 100 μL of the positive clone bacteria solution and add it to 10 mL of MG culture solution (containing 25 μg/mL rifampicin, 50 μg/mL kanamycin, pH=7.2) for cultivation, at 28°C, 180 rpm/min, shake the bacteria until OD600=0.6 -0.7, add an equal volume of 30% sterile glycerol, mix well, freeze quickly in liquid nitrogen, and store at -80°C for later use.

Example 1

In the embodiment of the present invention, the drought-resistant gene HvFRF1 was identified and cloned from the drought-resistant barley variety Hindmarsh. Hindmarsh is a drought-resistant and high-yield cultivar from Australia.

Cloning and bioinformatics analysis of HvFRF1 gene: Based on barley FRS gene family analysis and qRT-PCR analysis, a highly expressed FRF member under drought treatment was identified, and the full-length CDS region sequence of the gene was cloned from Hindmarsh, named HvFRF1 . The nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO.1.

The full length of the CDS region of HvFRF1 gene is 789bp, encoding a protein sequence of 262aa, the molecular weight of the protein is 31kDa, and the isoelectric point PI=9.2. The present invention also provides the protein encoded by the barley HvFRF1 gene, the amino acid sequence of which is shown in SEQ ID NO.2. The results of the prediction analysis of the functional domains of the HvFRF1 protein sequence showed that the protein contained a FAR1 functional domain. Therefore, the gene was classified as a member of FRF in the FRS gene family. The results of multiple sequence alignment showed that the HvFRF1 protein contained the conserved amino acids in the HMM logo diagram of the FAR1 functional domain. The phylogenetic tree analysis of the HvFRF1 protein sequence showed that the protein was located in the same clade as the three FRF members in rice.

Expression analysis of HvFRF1 gene: The research results showed that HvFRF1 gene was expressed in barley roots, stems and leaves, but the expression level was the highest in roots; the gene was significantly up-regulated by drought stress. The results of subcellular localization showed that the HvFRF1 gene was localized in the nucleus; the results of tissue localization showed that the HvFRF1 gene was mainly expressed in the epidermal cells and vascular bundles of the root system, and expressed in the vascular bundles of the leaves, indicating that the gene was related to water absorption and transportation.

Functional identification of the HvFRF1 gene: The full-length CDS of the HvFRF1 gene (with a stop codon) was connected into the overexpression vector super1300 by homologous recombination, and the Arabidopsis overexpression vector super1300:HvFRF1 of the HvFRF1 gene was constructed, and transformed into Arabidopsis Mustard Columbia, a stable transgenic line was obtained. The drought resistance of HvFRF1 transgenic Arabidopsis lines under drought treatment was significantly stronger than that of wild type.

Under drought conditions, the content of malondialdehyde (MDA) in HvFRF1 transgenic Arabidopsis lines was significantly lower than that of wild type, and the content of proline was significantly higher than that of wild type, indicating that overexpression of HvFRF1 gene in Arabidopsis can reduce membrane lipid peroxidation The degree and accumulation of the osmotic regulator proline enhances the drought resistance of plants. Through the Gateway method, the full-length CDS of the HvFRF1 gene (with a stop codon) was connected into the overexpression vector pBract214, and the barley overexpression vector pBract214:HvFRF1 of the HvFRF1 gene was constructed, and the barley Golden Promise was transformed to obtain a stable transgenic line.

Under drought treatment, the drought resistance of HvFRF1 transgenic barley lines was significantly stronger than that of wild type. Under drought treatment, the shoot dry weight of HvFRF1 transgenic barley lines was significantly higher than that of wild type, and they could maintain higher biomass.

Example 2

The invention uses the Australian drought-resistant and high-yield cultivar Hindmarsh as the main material to clone and analyze the key genes regulating the drought resistance of barley, which has important guiding significance for elucidating the molecular mechanism of barley responding to drought stress and barley drought-resistant breeding and production.

In the embodiment of the present invention, the cloning and analysis of the CDS region of the HvFRF1 gene include:

(1) Cloning of CDS region sequence of HvFRF1 gene

Based on the results of barley FRS gene family analysis and qRT-PCR expression analysis, a FRF member responding to drought stress was identified, and the full-length CDS region sequence of the gene (as shown in SEQ ID NO.1) was cloned from Hindmarsh, and its named HvFRF1.

Use the SteadyPure Plant RNA Extraction Kit (Aikerui, China) to extract total RNA from Hindmarsh leaves, and use DNaseI to remove genomic DNA contamination in the total RNA, and use the Evo M-MLV Plus 1st Strand cDNA SynthesisKit reverse transcription kit (Aikerui, China) China) reverse-transcribed the extracted total RNA into single-stranded cDNA. Design specific primers according to the sequence obtained by Blast, and the specific primer sequence is:

SEQ ID NO.3: HvFRF1-CDS-F:

5'-GGTTTTGTATGTTATGCAGATCGTGAGAC-3';

SEQ ID NO.4:

HvFRF1-CDS-R: 5'-CCAAACTGAATTGACTGTCCATGGTTG-3';

The CDS region of the gene was amplified using KOD OneTM PCR Master Mix-Blue (Toyobo, Japan). PCR system:

PCR program: first pre-denaturation at 94°C for 2 min, then denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 68°C for 5 s, denaturation annealing extension cycle 35 times, and extension at 68°C for 5 min. Connect the amplified product to the pEASY (full type gold, China) vector, transform Escherichia coli DH5α, pick positive clones and send them to the company for sequencing, and the sequenced correct ones are respectively extracted and stored in glycerol, and the resulting plasmid is named pEASY:HvFRF1 plasmid. PCR primer synthesis and gene sequencing were completed by Beijing Qingke Biotechnology Co., Ltd.

(2) Sequence analysis of HvFRF1 gene

The HvFRF1 protein sequence was predicted and analyzed by the SMART (https://smart.embl.de/) website for functional domains, and the results showed that the protein contained a FAR1 functional domain (Fig. 2A). It shows that HvFRF1 is FAR1-RELATED SEQUENCES-RELATED FACTOR (FRF). The conserved domain analysis of HvFRF1 protein sequence and Arabidopsis AtFRF1, AtFRF2, AtFRF3, and AtFRF4 protein sequences showed that the above protein sequences all contain the conserved amino acids of FAR1 (Figure 2B), and the same conserved amino acids can also be shown by the HMM logo. Amino acids, the larger the letter, the more conserved the amino acid.

FHY3 and FAR1 in Arabidopsis, 12 FRSs, 4 FRFs, 1 FRS, 3 FRFs in rice, and 1 FRS protein sequence in wheat were compared by Clustal W and MEGA 7 software A phylogenetic tree was constructed, and the results showed that HvFRF1 indeed belonged to the FRF subfamily, and was evolutionarily located in the same evolutionary branch as the FRFs of rice and Arabidopsis (Fig. 3A).

3. Expression of HvFRF1 gene in response to drought stress

Select plump and consistent barley Hindmarsh seeds to sow in 5L plastic culture pots with the same amount of nutrient soil, sow 6 seeds in each pot, pour the same amount of nutrient solution in each pot before sowing, and settle 4 plants in each pot 7 days after germination During this period, each pot was watered equally. Drought treatment was carried out at the stage of two leaves and one heart, and two treatments were set up: the control, in the whole process, each pot was watered in the same amount normally, and the soil moisture content was kept at 30-40%; the drought treatment, the watering was stopped to make the seedlings suffer from drought stress. The functional leaves of the control and drought-treated plants were taken 7 days and 14 days after watering was stopped to analyze the expression of HvFRF1 gene in response to drought stress.

The total RNA of each sample was extracted with the SteadyPure Plant RNA Extraction Kit (Aikerui, China), and the total RNA of each sample was extracted with the Evo M-MLV RT Kit with gDNA Clean for qPCRⅡ reverse transcription kit (Aikerui, China). The RNAs were reverse transcribed into single-stranded cDNAs, respectively. The expression of HvFRF1 gene in the corresponding samples was analyzed by qRT-PCR using SYBR green luciferase complex (Aikerui, China) and CFX ConnectReal-Time System PCR instrument (Bio-Red, USA), and an internal reference gene Actin was used to control the expression of HvFRF1 gene. The value is corrected. The PCR system is:

The PCR program was as follows: first, pre-denaturation at 94°C for 30 s, then denaturation at 95°C for 5 s, extension at 60°C for 30 s, and 40 cycles of denaturation and extension. The melting curve program is: 60°C-95°C, each step is 5s, and the increment is 0.5°C. Gene expression value changes were calculated using the 2 ^-ΔΔCq relative quantification method. Each experiment was repeated three times. The qRT-PCR primer sequences are:

SEQ ID NO.5: HvFRF1-qRT-PCR-F: 5'-CCCAAAACCCGCAACCTATTG-3';

SEQ ID NO.6: HvFRF1-qRT-PCR-R: 5'-GTAGGTATCCTGTCAGCGACC-3';

SEQ ID NO.7: Actin-F: 5'-TCGCTCCACCTGAGAGGAAG-3';

SEQ ID NO.8: Actin-R: 5'-GCTAGGATGGACCCTCCGAT-3';

The results of qRT-PCR showed that HvFRF1 gene was up-regulated by drought, and with the increase of drought stress, the induced up-regulated expression was higher (Figure 3B), indicating that HvFRF1 gene responded to drought stress.

(3) Subcellular localization of HvFRF1 gene

Using the saved pEASY:HvFRF1 plasmid as a template, a pair of primers HvFRF1-super promoter-GFP-F and HvFRF1-super promoter-GFP-R were designed to amplify the ORF fragment of the HvFRF1 gene (remove the stop codon TAA) for subcellular positioning analysis. The primer sequence is (the underlined part is the restriction site):

SEQ ID NO.9: HvFRF1-super promoter-GFP-F: 5'-CCAAATCGACTCTAG TCTAGA ATGCCGGAGGTGATGTCCA-3';

SEQ ID NO.10: HvFRF1-super promoter-GFP-R: 5'-GCCCTTGCTCACCAT GGTACC TACCAATAAACGGGCACAAGG-3';

HD Cloning Kit同源重组试剂盒(Takara，日本)将胶回收纯化的ORF片段与super1300-super promoter-GFP载体进行同源重组反应。将反应产物转化大肠杆菌DH5α，阳性克隆送公司测序，测序正确的单克隆(确保无移码，能翻译成完整的基因-GFP融合表达蛋白)提质粒保存，所得质粒为super promoter-HvFRF1-GFP(SEQ ID NO.27)。use

The HD Cloning Kit Homologous Recombination Kit (Takara, Japan) performed homologous recombination reaction on the ORF fragment recovered and purified from the gel with the super1300-super promoter-GFP vector. The reaction product was transformed into Escherichia coli DH5α, the positive clone was sent to the company for sequencing, and the sequenced correct single clone (to ensure no frame shift and can be translated into a complete gene-GFP fusion expression protein) was stored as a plasmid, and the obtained plasmid was super promoter-HvFRF1-GFP (SEQ ID NO. 27).

Get the leaves of 3-4 weeks old Arabidopsis thaliana and place them in a 50ml Erlenmeyer flask, add 5-10ml enzymatic hydrolysis solution (1.5% Cellulase R10, 0.75% Macerozyme R10, 0.6M Mannitol, 10mM MES, 40% 2-Hydroxy-1-ethanethio ) to soak all the leaves, wrap them with a parafilm after vacuuming, and enzymatically hydrolyze at 23° C. for 3 hours under weak light. Use 40μm nylon gauze to filter the enzymatic hydrolysis product, collect the protoplasts into a 2ml centrifuge tube, centrifuge at 400rpm/min at 4°C for 5min to collect the precipitate, and use pre-cooled W5 solution (154mM NaCl, 125mM CaCl ₂ , 2mM KH ₂ PO ₄ , 2mM MES) was washed twice, resuspended and left to stand on ice for 30 min. Centrifuge the suspended suspension at 400rpm/min for 5min, add MMG solution (0.4M Mannitol, 15mMMgCl ₂ .6H ₂ O, 4mM MES) to resuspend the pellet, and add super promoter-HvFRF1-GFP plasmid and nuclear localization marker as needed Plasmid and 40% PEG4000 solution, mixed by inversion slowly, 22.5 ℃ water bath for 15-20min. Add a certain volume of W5 solution to dilute the protoplasts to terminate the reaction, centrifuge at 400rpm/min for 5min, wash twice with W5 solution and resuspend, and culture overnight at 23°C under low light. The protoplast suspension cultured overnight was centrifuged at 400 rpm/min for 5 min to collect the protoplasts, and the fluorescence distribution was observed under a confocal laser microscope (LSM780, Carl Zeiss, Germany).

The results showed that the HvFRF1 gene was localized in the nucleus and overlapped with the signal of the nuclear localization marker ( FIG. 4B ).

(4) Tissue localization of HvFRF1 gene

Hindmarsh seeds were sterilized with 2% H ₂ O ₂ for 30min, then washed 5-6 times with distilled water, placed the sterilized seeds in the germination box and cultured in the dark in the growth room (22°C/18°C) until germination, germination Then add light (22℃/18℃, day/night). On the 7th day, select seedlings with consistent growth and transfer them to a 5L black plastic bucket filled with barley basic culture solution (1/5 Hogland formula, PH=5.8-6.0), cover with a 9-hole plastic cover, and use 1 seedling per hole. The sponges were fixed and cultured in a barley culture room. The whole culture process was ventilated with an aeration pump, and the culture medium was replaced every 5 days. The roots, stems and leaves of the plants were taken at the three-leaf stage and the tillering stage, respectively, for tissue expression analysis of the HvFRF1 gene. RNA extraction, reverse transcription, primer sequences, qRT-PCR system and procedures are the same as those described in the section "3. HvFRF1 Gene Expression in Response to Drought Stress".

The results showed that the HvFRF1 gene was expressed in the roots, stems and leaves of the plants, but the expression level in the roots was higher ( FIG. 4A ).

Take the Hindmarsh roots and leaves hydroponic at the three-leaf stage, cut out tissue fragments of about 1 cm respectively, put them into fresh formaldehyde fixative (63% Ethanol, 5% acetic acid, 2% formaldehyde) immediately and vacuumize. Wash Buffer 1 (63% Ethanol, 5% glacial acetic acid) three times, and embed leaves and roots with 5% agarose and 5% low-melting point agarose, respectively. Slice the embedded tissue (50-80 μm) with a vibrating microtome, put each cut tissue sample into a PCR tube containing RNase inhibitor (Takara, Japan), and digest it with RNase free DNase I (Takara, Japan) at 37°C overnight. PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, Japan) and In situHvFRF1-R primers were used for reverse transcription for In situ PCR research. The 18S internal reference gene was used as a positive control, and no primer was added during reverse transcription as a negative control. In situ PCR primer sequences are:

SEQ ID NO.11: In situ HvFRF1-F:

5'-AACACTACAAACTGTATGCG-3';

SEQ ID NO.12: In situ HvFRF1-R:

5'-GTTCTAGATTGCACGAATGA-3';

SEQ ID NO.13: In situ Hv18S-F:

5'-GGTAATTCCAGCTCCAAT-3';

SEQ ID NO.14: In situ Hv18S-R:

5'-GTTTATGGTTGAGACTAG-3';

Using each tissue section as a template, the gene was amplified with digoxin-labeled dUTP (Roche, Germany), PCR system:

PCR program: first pre-denaturation at 95°C for 2 min, then denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 68°C for 5 s, denaturation annealing extension cycle 30 times, and extension at 68°C for 5 min. The tissue sections after PCR were washed twice with 1×PBS buffer (10mM Na ₂ HPO ₄ , 130mM NaCl, pH=7.5.), added 1×Block stock solution (10mg BSA dissolved in 1mL 10xPBS), and kept on ice for 30min. Add Anti-DIG-AP antibody (Roche, Germany), incubate at 37°C for 1 h, then add BM-Purple (Roche, Germany) for color reaction, wash and fix with 40% (v/v) glycerol, spread on slide glass On the slide, the tissue staining of the HvFRF1 gene was observed under a stereomicroscope.

The results showed that the blue color mainly appeared in the epidermal cells of roots and the vascular bundles of roots and leaves, indicating that the HvFRF1 gene was mainly expressed in these tissue parts, and the epidermal cells and vascular bundles were closely related to the water absorption and transportation of plants, indicating that the HvFRF1 gene The expression of is beneficial to the absorption and utilization of water by barley.

Example 3

In the embodiment of the present invention, the functional verification of HvFRF1 gene includes:

(1) super1300: HvFRF1 vector construction

Using the saved pEASY:HvFRF1 plasmid as a template, a pair of primers HvFRF1-OE-F1 and HvFRF1-OE-R1 were designed to amplify the ORF fragment of HvFRF1 gene. The primer sequences are:

SEQ ID NO.15: HvFRF1-OE-F1: 5'-CGACTCTAGTCTAGAAAGCTTATGCCGGAGGTGATGTCC-3';

SEQ ID NO.16: HvFRF1-OE-R1: 5'-CGATCGGGGAAATTCGAGCTCTTATACCAATAAACGGGC-3';

HD Cloning Kit同源重组试剂盒(Takara，日本)将胶回收纯化的ORF片段与superl300载体(本实验室保存)进行同源重组反应。将反应产物转化大肠杆菌DH5α，阳性克隆送公司测序，测序正确的单克隆提质粒保存，所得质粒为superl300:HvFRF1(图5A)。use

HD Cloning Kit Homologous Recombination Kit (Takara, Japan) performed homologous recombination reaction on the ORF fragment recovered and purified from the gel and the super1300 vector (preserved in our laboratory). The reaction product was transformed into Escherichia coli DH5α, the positive clone was sent to the company for sequencing, and the sequenced correct single clone was extracted and stored as a plasmid, and the obtained plasmid was super1300:HvFRF1 (Figure 5A).

The above plasmids were transformed into Agrobacterium competent cells EHA105, coated with rifampicin+kanamycin-resistant YEB plates, cultured at 28°C for about 40-48 hours, and positive clones were verified by PCR. Add an equal volume of 30% sterile glycerol to the positive cloning bacteria solution, mix well, and then quickly freeze in liquid nitrogen and store at -80°C for later use.

(2) Arabidopsis genetic transformation and positive plant verification:

Inoculate the Agrobacterium containing the super1300:HvFRF1 plasmid into 5 ml of liquid LB medium containing rifampicin + kanamycin antibiotics, and shake the bacteria at 220 rpm/min overnight (about 18 hours) at 28°C under dark culture conditions. According to the ratio of 1:100, take 1ml of overnight bacteria, inoculate in 100ml of liquid LB culture medium containing rifampicin + kanamycin antibiotics, culture in dark at 28°C, 220rpm/min until OD value = 1.2-2.0 , pour into a sterilized 50ml centrifuge tube, centrifuge at 5000rpm/min, 28°C for 8min, collect the bacteria, and discard the supernatant culture solution. Resuspend the collected cells with transformation permeate (1/2MS, 5% sucrose, 20% silwet L-77, 0.05% MES, 1% AS, 1% 6BA) to make the OD600 value 0.8-1.0. Soak the flower buds of wild-type Arabidopsis thaliana (Columbia type) at the flowering stage in the above-mentioned osmotic solution for 1-2 hours. After the infection, the plants were placed sideways in a moist tray, kept in the dark for 48 hours, and then cultured upright (23°C, 16/8h, day/night). Collect seeds as much as possible at last, and the collected seeds are the T1 generation. After the seeds of the T1 generation were sterilized, spread a large number of plates on a plate containing 25mg/L hygromycin, and select seedlings with good root growth and strong growth and transferred them to the nutrient bowl (nutrient soil: vermiculite: pearl salt = 6:3:1) to continue nourish. Leaf DNA was extracted before bolting for positive plant verification. The superl300:HvFRF1 plasmid was used as a positive control, and wild-type Arabidopsis was used as a negative control. For the plants that were verified as positive, the leaf RNA was extracted and reverse-transcribed, and the expression level was analyzed, and Actin of Arabidopsis was used as an internal reference gene. Positive plant verification primers and expression analysis primer sequences are:

SEQ ID NO.17: super1300-F:

5'-GCTCGCGGTGACGCCATTTCGCCTTTTC-3';

SEQ ID NO.18: HvFRF1-R1:

5'-GTGTAACCAATGGCTTCACGTTGGCCATG-3';

SEQ ID NO.19: AtActin-F:

5'-AGGAAGGATCTGTACGGTAAC-3';

SEQ ID NO.20: AtActin-R:

5'-TTCTGTGAACGATTCCTGGAC-3';

Positive plants with high HvFRF1 gene expression were selected for harvesting, and T2 seeds were obtained. Take T2 seeds and spread them on a plate containing 25mg/L hygromycin, record the resistance performance according to the seedling rate, and keep the lines with positive seedling:negative seedling segregation ratio of 3:1 for use. Select robust seedlings from the culture dishes of the strains used in the T2 generation and transfer them to nutrient pots for further cultivation, and harvest T3 seeds from individual plants after maturity. Take 100 seeds from each of the T3 generation lines and spread them on a plate containing 25 mg/L hygromycin, record the resistance performance according to the seedling rate, and keep the lines that meet the chi-square test of 95:5.

(3) Identification of drought resistance phenotypes of Arabidopsis lines overexpressing HvFRF1 gene:

The seeds of wild-type Arabidopsis thaliana and overexpression strains OE1, OE2 and OE3 were sterilized with 75% alcohol for 2 minutes, then 10% sodium hypochlorite was added, sterilized for 5 minutes, and washed 4-5 times with sterilized water, 2 minutes each time. Spread the sterilized seeds evenly on 1/2 MS solid medium (containing 1.5% sucrose, 0.4% plant gel, pH 5.8), and absorb excess water on the surface of the medium. Put the 1/2 MS culture dish sown with seeds at 4°C for vernalization for 3 days, then place it in the culture room of Arabidopsis thaliana (23°C, 16/8h, day/dark), and transplant it in the four-leaf stage In the nutrition bowl. Drought treatment was carried out 15 days after transplanting, and two treatments were established: control, in the whole process, each pot was watered in the same amount normally, and the soil moisture content was kept at 30-40%; drought treatment, watering was stopped to make the seedlings suffer from drought stress. The drought-resistant phenotypes of the plants were observed on the 12th day after watering was stopped, and the functional leaves of the control and drought-treated plants were taken to measure the physiological indicators.

The results showed that under control conditions, there was no significant difference in the growth of wild-type and overexpressed Arabidopsis thaliana, but the growth of overexpressed plants was significantly better than that of wild-type plants after drought stress, showing a smaller degree of wilting (Figure 5B). After drought treatment, the MDA content of wild-type Arabidopsis was significantly increased, while that of the overexpression line was significantly decreased (Fig. 5C). In addition, the accumulation of proline in the overexpression line was significantly higher than that in the wild type (Fig. 5D).

The above results indicated that the expression of HvFRF1 gene can reduce the degree of membrane lipid peroxidation and accumulate the osmotic regulator proline to enhance the drought resistance of plants.

Example 4

The embodiment of the present invention provides a pBract214:HvFRF1 vector construction method:

The Gateway method was used to construct the overexpression vector of HvFRF1 gene, namely pBract214:HvFRF1. Using the saved pEASY:HvFRF1 plasmid as a template, a pair of primers HvFRF1-OE-F2 and HvFRF1-OE-R2 were designed to amplify the ORF fragment of HvFRF1 gene. The primer sequences are:

SEQ ID NO.21: HvFRF1-OE-F2:

5'-GGGGACAAGTTTGTACAAAAAAAGCAGGCTTCACCATGCCGGAGGTGATGTCCACGCC-3';

SEQ ID NO.22: HvFRF1-OE-R2:

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTGTTATACCAATAAACGGGCACAAG-3';

The PCR amplification products were detected by 1% agarose gel electrophoresis, the target products were recovered and purified by gel, and the concentration of the recovered products was measured. Subsequently, Gateway BP Clonase II enzyme mix (Invitrogen, USA) was used to perform BP recombination reaction with the gene recovery product of known concentration and the pDONR (Zeo) entry vector. BP recombination reaction system:

React at 25°C for 4 hours, add 1 μL of Proteinase K, continue to react at 37°C for 10 minutes, the reaction product is immediately transformed into E. coli DH5α competent cells, coated with bleomycin-resistant LB solid medium, cultured at 37°C for about 16 hours, picked Monoclonal, shake the bacteria for bacterial fluid PCR, send the bacterial fluid of positive clones to the company for sequencing, the sequenced correct single clone is multiplied, the bacterial fluid is stored in glycerol and the extracted plasmid is preserved, and the plasmid is named pDONR(Zeo):HvFRF1.

Gateway LR Clonase II enzyme mix (Invitrogen, USA) was used to carry out LR recombination reaction between the above-mentioned known concentration of the plasmid and the target vector pBract214 (SEQ ID NO.28) (provided by Dr. Peter Dominy, University of Glasgow, UK). LR recombination reaction system:

React at 25°C for 4 hours, add 1 μl of Proteinase K, continue to react at 37°C for 10 minutes, the reaction product is immediately transformed into E. coli DH5α competent cells, coated with kanamycin-resistant LB solid medium, cultured at 37°C for about 16 hours, picked Monoclonal, shake the bacteria to carry out bacterial liquid PCR, send the bacterial liquid with the target band to the company for sequencing, the sequenced correct single clone is multiplied, the bacterial liquid is stored in glycerol and the extracted plasmid is stored, and the plasmids are respectively named pBract214:HvFRF1 (Fig. 6A).

The correctly sequenced pBract214:HvFRF1 plasmid and pSoup plasmid were co-transformed into Agrobacterium competent cell AGL1, coated with rifampicin+kanamycin-resistant YEB plate, cultured at 28°C for about 40-48h, and PCR verified positive clones. Take 100 μL of the positive clone bacteria solution and add it to 10 mL of MG culture solution (containing 25 μg/mL rifampicin, 50 μg/mL kanamycin, pH=7.2) for cultivation, at 28°C, 180 rpm/min, shake the bacteria until OD600=0.6 -0.7, add an equal volume of 30% sterile glycerol, mix well, freeze quickly in liquid nitrogen, and store at -80°C for later use.

Example 5

The embodiment of the present invention provides a method for genetic transformation of young barley embryos comprising:

The immature embryos of cultivated barley Golden Promise (Golden Promise) 2-3 weeks after flowering were selected as genetic transformation materials. The grains meeting the standards were stripped from the ears, and the awns were removed. The seeds were surface-sterilized with 70% alcohol for 30 seconds, and washed several times with sterilized water. Then soak it in 10% sodium hypochlorite for 4 minutes, and wash it several times with sterilized water. On sterile filter paper, separate the immature embryos, remove the hypocotyl, put the scutellum on the callus induction medium, and culture in the dark at 23-24°C for 2-3 days. Add 400 μL of the preserved Agrobacterium liquid into 10 mL of MG liquid medium without antibiotics, shake the bacteria at 28°C and 180 rpm/min until OD600=1.3-1.4, and use for immature embryo infection. The prepared Agrobacterium infection solution was dripped on each immature embryo and allowed to dry. Seal the plate with parafilm and co-culture in the dark at 23-24°C for 2 days. Transfer the immature embryos to a fresh callus induction medium plate for selection culture (containing 50 mg/L hygromycin, 160 mg/L Temimycin). After dark culture at 23-24°C for 56 days (replace the medium plate every 14 days), transfer the callus isolated from the immature embryos to the transfer medium, and culture for 21 days at 24°C under low light, at this time green point. Transfer the green dots to the subculture medium to continue culturing. When the upper leaves reach 2-3cm, the roots begin to build. The seedlings were transferred to rooting medium without any growth regulators and antibiotics unchanged. Afterwards, the seedlings with established roots were taken out, the culture medium was washed off, transferred to plastic culture pots containing vermiculite and nutrient substrate, and grown in an artificial climate chamber (22°C/18°C, day/night) until the seeds were harvested.

In the embodiment of the present invention, the verification of transgenic plants and the phenotypic identification of HvFRF1 overexpression barley lines include:

DNA was extracted from leaves of transgenic plants at the seedling stage by CTAB method. Wild-type Golden Promise was used as a negative control and pBract214-HvFRF1 plasmid was used as a positive control to verify whether the vector carrying the target fragment was transferred into the barley genome. Select plants with positive DNA verification, extract leaf RNA and reverse transcribe, take the expression level of HvFRF1 gene in wild-type Golden Promise as a reference, verify the expression level of HvFRF1 gene by fluorescent quantitative PCR, and select two expression levels to compare tall lines OE1 and OE2 (Fig. 6C). The qRT-PCR primers for verifying the expression of positive plants are the same as the HvFRF1-qRT-PCR-F, HvFRF1-qRT-PCR-R, Actin-F and Actin-R primers in the section "Expression of HvFRF1 Gene in Response to Drought Stress". Verify that the DNA amplification primer sequences of positive plants are:

SEQ ID NO.23: pBract214-HvFRF1-F: 5'-ACAAGTTTGTACAAAAAAGCAGGCTTC-3';

SEQ ID NO.24: pBract214-HvFRF1-R:

5'-TTATACCAATAAACGGGCACAAG-3';

The soil culture drought stress experiment was carried out on barley wild-type Golden Promise and stable HvFRF1 overexpression lines at the three-leaf stage. The growth of the expressing plants was significantly better than that of the wild type, showing less wilting (Fig. 6B) and greater biomass accumulation (Fig. 6D), indicating that the HvFRF1 gene positively regulates the drought resistance of barley.

In summary, through the isolation, cloning and analysis of the barley HvFRF1 gene, combined with heterologous and homologous overexpression techniques, the function of the gene was verified on barley GP. The results showed that the overexpression of HvFRF1 significantly enhanced the drought resistance of plants. The invention provides a theoretical basis and related genes for drought-resistant breeding and production of barley.

Among them, the super1300 vector sequence SEQ ID NO.25, wherein the sequence of the multiple cloning site includes: TCTAGAAAGCTTCTGCAGGGGCCCGGGGTCGACATTTAAATACTAGTGGATCCGGTACCGAGCTC;

The specific sequence is as follows:

GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGC CTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGG ATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTAGTAG GATCCGCGAGCTGCTCCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTTTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCCAACTTTTTTCTTGATCCGCAG CCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACCAATCGACTCT AGTCTAGAAAGCTTCTGCAGGGGCCCGGGGTCGACATTTAAATACTAGTGGATCCGGTACCGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGG GTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAAC AAAGGGTAATATCGGGAAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTAAAGCAA GTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCCAAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATC GTTCAAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACGTCT CTCTTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCGGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCC GATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGG CCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGC GGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGC ATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGG GCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAATTTCTAATTCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAAT TAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACCCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCG ATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAAAAGCTGTTTTCTGGTATTTAAGGTTTTTAGAATGCAAGGA ACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTAAAAATGACGGA CAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGC ATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTTCCCACGGCGACCTGGGAGA CAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTACT GTACCTAGAATGCATGACCAAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAACTG TCCTTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTTCTTTCCTGCGTTATTCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTTCTCTTACGCAT CTGTGCGGTATTTCACACCCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAG GTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAG CTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTAT CCACAGGAAAGAGACCCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAAC TCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCC GCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTTTCTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGG CCGGGCTTGTCTCCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGAT GCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGC GGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATT TTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTT GCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCA AGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGG ACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGG GCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTTATTTACTCATCGCATCATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCC GCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGT CAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCG GGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGG CCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCAGCAGGCCAGCGGCGGC GCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATC CCGAGGGGAACCCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTG GGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC.

super1300: HvFRF1 carrier sequence (restriction site in red, HvFRF1 gene sequence in blue) SEQ ID NO.26, wherein, restriction site includes: AAGCTT, GAGCTC; HvFRF1 gene sequence is: atgccggaggtgatgtccacgccacagcaacccacagccatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcga ggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagc gaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtga agccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtt tttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataa;

The specific sequence of SEQ ID NO.26 is:

GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGC CTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGG ATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTAGTAG GATCCGCGAGCTGCTCCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTTTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCCAACTTTTTTCTTGATCCGCAG CCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACCAATCGACTCT AGTCTAGAAAGCTTatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttga agatcaagaggaaagtagagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctga agaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagt caacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttatggtataaGAGCTCGAATTTCCCCGATC GTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATTCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGC GGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAAT CGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTG GCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACTCGTCACTCCAAGAATATCAAAGATACAGTCAGAAGACCAAAGGGC TATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCACACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAAC CACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTAAAATCTTCTCTCTCGAGCTTTCGCAGATCCGGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGAC GTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTG GGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGC GCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGG GGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCG ACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATA AGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTAACACCACAATATC CTGCCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATT GTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGGTTTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAA ATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGT TCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGTCCCTACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACT GAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTAT GACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAATGCATGACCAAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAGATCAAAGGATCTTCTT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCG GCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGC GTTATCCCCTGATTCTGTGGATAACCGTATTACCCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTACGCATCTGTGCGGTATTTCACACGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTA TCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGC GGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGC GGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGAGACCTTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCG GATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGC GGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGG CCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCG TAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCC TAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACG CCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATTTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAG ATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGA TTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACA AAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGT CAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTC CCCTTGCGTATTTGTTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTAGACGGCGGCGCTCGGTTTCTTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCC GGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAG GGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAAC ACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTC GCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAG GAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTT ATTCTAATAAACGCTCTTTTTCCTTAGGTTTACCCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTT GGGTAACGCCAGGGTTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC.

super promoter-HvFRF1-GFP SEQ ID NO.27, wherein the restriction site sequence includes: TCTAGA, GGTACC, HvFRF1 gene sequence is:

atgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagag gaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaac aacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagctt gataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgttttggta;

The GFP gene sequence is:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCCTGACCTACGGCGT GCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAG CTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA;

The specific sequence of SEQ ID NO.27 is:

GGATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGC CTGTAGGATCCCTGAAAGCGACGTTGTGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTGGTGGACCCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTGTAGG ATCCCTGAAAGCGACGTTGGATGTTAACATCTACAAATTGCCTTTTCTTATCGACCATGTAACGTAAGCGCTTACGTTTTTGGTGGACCTTGAGGAAACTGGTAGCTGTTGTGGGCCTGTGGTCTCAAGATGGATCATTAATTTCCACCTTCACCTACGATGGGGGCATCGCACCGGTGAGTAATATTGTACGGCTAAGAGCGAATTTGGCCTAGTAG GATCCGCGAGCTGCTCCAATCCCATTGCTTTTGAAGCAGCTCAACATTGATCTCTTTCTCGATCGAGGGAGATTTTTCAAATCAGTGCGCAAGACGTGACGTAAGTATCCGAGTCAGTTTTTTTTTTCTACTAATTTGGTCGTTTATTTCGGCGTGTAGGACATGGCAACCGGGCCTGAATTTCGCGGGTATTCTGTTTCTATTCCCAACTTTTTTCTTGATCCGCAG CCATTAACGACTTTTGAATAGATACGCTGACACGCCAAGCCTCGCTAGTCAAAAGTGTACCAAACAACGCTTTACAGCAAGAACGGAATGCGCGTGACGCTCGCGGTGACGCCATTTCGCCTTTTCAGAAATGGATAAATAGCCTTGCTTCCTATTATATCTTCCCCCAAATTACCATACATTACACTAGCATCTGAATTTCATAACCAATCTCGATACCAATCGACTCT AGTCTAGAatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatca agaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaag aacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcaaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacag cttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtaGGTACCATGGTGAGCAAGGGCGAG GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTCGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGA GTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATGTAATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATT AGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC ACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCAGAAGACCAAAGGGCTATTGAGACTTTTCAAACAAAGGGTAATA TCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCCAAAGATGGACCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTTCAAAGCAAGTGGATTGAT GTGATAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGC CTCTGCCGACAGTGGTCCCAAAGATGGACCCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTTATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCCTCTACAAAT CTATCTCTCTCGAGCTTTCGCAGATCCGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTC TACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATCGCTGCGCCGATCTTAGCC AGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGC TCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTT GACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAGAAATA GAGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTAATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATTCGGCGT TAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAAATTTGTTTACACCCACAATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCGCAGACTTTGCTCATGTTACCGATGCTATTCG GAAGAACGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGATACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAAGCTGTTTTCTGGTATTTAAGGTTTTGAATGCAAGGAACAGTGAATT GGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGTATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTAAAAATGACGGACAGCCGGTAT AAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTT TCACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCGAAGAGGAACTTGTCTTTTTCCCACGGCGACCTGGGAGACAGCAACATCT TTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTACTGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTAGTACCTAGAAT GCATGACCAAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGT AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTTCTTTCCTGCGTTATTCCCCTGATTCTGTGGATAACCGTATTACCCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTCTCCTTACGCATCTGTGCGGTAT TTCACACCCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCG TCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTTCGGCT GTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAAGAGTTTTAATAAGTTTTAAAGAGTTTAGGCGGAAAAATCGCCTTTTTTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAGA GACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCG CTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTC GCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGT CTCCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTA TCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACT TCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGC CTCCTTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGCTGCTGCGT GCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGG CAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGT CCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCT AGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTTATTTACTCATCGCATCATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGC ATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTC ACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGC GGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGCCAGGCGGTC TAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCAGCAGGCCAGCGGCGGCGCTCTTGTTCAT GGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTACTTTGATCCCGAGGGGA ACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTAAATATCCGCCCTTTTAAATATCCGTTATTCTAATAAACGTCTTTTTCTCTAGGTTTACCCGCCAATAATATCCTGTCCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGA TCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCC.

pBract214 vector SEQ ID NO.28, the sequence of the attR arm includes: ACAAGTTTGTACAAAAAAGCTGAAC, GTTCAGCTTTCTTGTACAAAGTGG;

The specific sequence of SEQ ID NO.28 is:

TTTTTATCCCCGGAAGCCTGTGGATAGAGGGTAGTTATCCACGTGAAACCGCTAATGCCCCGCAAAGCCTTGATTCACGGGGCTTTCCGGCCCGCTCCAAAAACTATCCACGTGAAATCGCTAATCAGGGTACGTGAAATCGCTAATCGGAGTACGTGAAATCGCTAATAAGGTCACGTGAAATCGCTAATCAAAAGGCACGTGAGAACGCTAATAGCCCTTT CAGATCAACAGCTTGCAAACCCCCTCGCTCCGGCAAGTAGTTACAGCAAGTAGTATGTTCAATTAGCTTTTCAATTATGAATATATATCAATTATTGGTCGCCCTTGGCTTGTGGACAATGCGCTACGCGCACCGGCTCCGCCCGTGGACAACCGCAAGCGGTTGCCCACCGTCGAGCGCCAGCGCCTTTGCCCACAACCCGGCGGCCGGCCGCAACAGA TCGTTTTATAAATTTTTTTTTTTGAAAAAGAAAAAGCCCGAAAGGCGGCAACCTCTCGGGCTTCTGGATTTCCGATCCCCGGAATTAGATCTTGGCAGGATATATTGTGGTGTAACGTATCACAAGTTTGTACAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTAGACTCGACGCGTCCTAGAGATCCGTCAACATGGTGGAGCACGACACTCTCGTCTAC TCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCC ACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTCATTTCATTTGGAGAGGACGACCCCGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAA GTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATT GCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTTATT TCGTGTGTCTATGATGATGATGATAACTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTC GCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAG CAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCG GCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGATCCGGAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAG CATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCATCGATGATATCAGATCAAGGGTGGGCGCGCCGAACCAGCTTTCTTGTACAAAGTGGT GATCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTTTTATCTATCTTTATACATATTTAAACTTTACTCTACGAATAATAATAATCTATAGTACTACAATAATATCAGT GTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTTATCTTTTTAGTGTGCATGTGTTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATAATAACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTATAGACTAATTTTTTAGTACATCTATTTTATTCTATTTTAGC CTCTAAATTAAGAAAAAACTAAAACTCTATTTTAGTTTTTTTTATTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGA AGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGTCGGACGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCC GTAATAAATAGACACCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACAACCAGATCTCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTACCTTCTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTT GTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCG GGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTAATTCCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGG GTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTG ATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATAACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCCACC CTGTTGTTTGGTGTTACTTCGCCCATCACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATTAAATTAGATTTTGCATAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGAT CCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCC GCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGG GTTTATTGAGAATATGTTTTTCGTCTCAGGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAG TACTGCGATGAGTGGCAGGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCC GGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGAT ATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCAT TAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTACGTTTCTCGTTCAGCTTGTACAAAGTGGTGATGGGGGATCCACTAGTTCTAGAATTCGA TTGAGTCAAGCAGGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAATATAGCGCGCAAACTAG GATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGACCGGCATGCAAGCTGATATCAATCACTAGTGAATTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTAT CAGCTCACTCAAAGGCGGTAATACGGTTATTCCACAGAATCAGGGGATAACGCAGGAAAGAACATGAAGGCCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTGAGATCTCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA AATCGACGCTCCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAGAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA AAATGAAGTTTTAAATCAATCTTAAAGTATATGTGTAACATTGGTCTAGTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTTCATATCAGGATTATCAATACCATATTTTGAAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATAC ACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTCACCTGAATTCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCTGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTTCATCTGTAAAACATTGGCAAC GCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGGTAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTTGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCTTGTATTACTGTTTTATGTAAGCAGACAGTTTTTTGTGT TCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATG ATGGGGCGATTCAGGCGATCCCCATCCAACAGCCGCCGTCGAGCGGGCT.

pBract214: HvFRF1 vector SEQ ID NO.29, the sequence of the attB arm is: ACAAGTTTGTACAAAAAAGCAGGCTTCACC, CACCCAGCTTTCTTGTACAAAGTGGT;

The sequence of the HvFRF1 gene sequence is:

atgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtgaactgataagagcatcacttgtctgccatagagctgggttgaagatcaagag gaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaatggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaac aacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatacactgatatgcaggacacgattgagtacttcaaaagcagtcaacagctt gataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttgtgcccgtttattggtataa

The specific sequence of SEQ ID NO.29 is:

TTTTTATCCCCGGAAGCCTGTGGATAGAGGGTAGTTATCCACGTGAAACCGCTAATGCCCCGCAAAGCCTTGATTCACGGGGCTTTCCGGCCCGCTCCAAAAACTATCCACGTGAAATCGCTAATCAGGGTACGTGAAATCGCTAATCGGAGTACGTGAAATCGCTAATAAGGTCACGTGAAATCGCTAATCAAAAGGCACGTGAGAACGCTAATAGCCCTTT CAGATCAACAGCTTGCAAACCCCCTCGCTCCGGCAAGTAGTTACAGCAAGTAGTATGTTCAATTAGCTTTTCAATTATGAATATATATCAATTATTGGTCGCCCTTGGCTTGTGGACAATGCGCTACGCGCACCGGCTCCGCCCGTGGACAACCGCAAGCGGTTGCCCACCGTCGAGCGCCAGCGCCTTTGCCCACAACCCGGCGGCCGGCCGCAACAGA TCGTTTTATAAATTTTTTTTTTTGAAAAAGAAAAAGCCCGAAAGGCGGCAACCTCTCGGGCTTCTGGATTTCCGATCCCCGGAATTAGATCTTGGCAGGATATATTGTGGTGTAACGTATCACAAGTTTGTACAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACCTAGACTCGACGCGTCCTAGAGATCCGTCAACATGGTGGAGCACGACACTCTCGTCTAC TCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCC ACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATAAGGAAGTTCATTTCATTTGGAGAGGACGACCCCGATATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAA GTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATT GCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGGTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTTATT TCGTGTGTCTATGATGATGATGATAACTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTC GCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAG CAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCG GCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGGGATCCGGAGAGCTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAG CATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTCATCGATGATATCAGATCAAGGGTGGGCGCGCCGAACCAGCTTTCTTGTACAAAGTGGT GATCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTTTTATCTATCTTTATACATATTTAAACTTTACTCTACGAATAATAATAATCTATAGTACTACAATAATATCAGT GTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTTATCTTTTTAGTGTGCATGTGTTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATAATAACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTATAGACTAATTTTTTAGTACATCTATTTTATTCTATTTTAGC CTCTAAATTAAGAAAAAACTAAAACTCTATTTTAGTTTTTTTTATTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGA AGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGTCGGACGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCC GTAATAAATAGACACCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACAACCAGATCTCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTACCTTCTCTAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTT GTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCG GGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTAATTCCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGG GTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTG ATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATAACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCCACC CTGTTGTTTGGTGTTACTTCGCCCATCACAAGTTTGTACAAAAAAAGCAGGCTTCACCatgccggaggtgatgtccacgccacagcaacccacagccatgatgagatttgacactcttgaggatgctgagaaacactacaaactgtatgcgaggcagaaaggttttggagtaaggtattgtttccgaaaaaggtcagaggctagtggtga actgataagagcatcacttgtctgccatagagctgggttgaagatcaagaggaaagtagacacccaaaacccgcaacctattgcccctgagaggagtaggaatacaactgaaagaacaaactgcccagcccgtatgtttgtgaagcgaagagataatgcctgggttgtaacagaaataaatgacaaccataaccaccctctcataaagaaat ggtcgctgacaggatacctacgatcacatagacatatccctgaagaagaacaacagtttgtgaagttgcttcattcgtgcaatctagaaccttctagacagatgcagttgttgacagagttgcatggccaacgtgaagccattggttacactgacaaagacttggcaaatttgctagcaaagttccgggctgagcacaaatac actgatatgcaggacacgattgagtacttcaaaagcagtcaacagcttgataaagactttttttacaagtacaagcttgatgatgaaaataaggtccaatgcatttactggattgatggttcagcgagaagggcttacaagtttttcagtgattgtgtctcttttgacacaacatacttgaccaatatatacaagatgccttg tgcccgtttatggtataaCACCCAGCTTTCTTGTACAAAGTGGTGATGGGGGATCCACTAGTTCTAGAATTCGATTGAGTCAAAGCAGGATCGTTCAAACATTTGGCAATAAAGTTTCTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATGACATGTAATGCATGACGTT ATTTATGAGATGGGTTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAAATAGCGCGCAAACTAGGATAAATTATCGCGCGGTGTCATCTATGTTACTAGATCGACCGGCATGCAAGCTGATATCAATCACTAGTGAATTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCTTTTGTTCCTTAGTGAGGGTTAATTGCGCGC TTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCG GTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCCAGAATCAGGGGATAACGCAGGAAAGAACATGAAGGCCTTGACAGGATATATTGGCGGGTAAACTAAGTCGCTGTATGTGTTTGTTTGAGATCTCATGTGAGCAAAGG CCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTCGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAGAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCCAGAAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTTCTACGGGGTCTGAC GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGTGTAACATTGGTCTAGTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTTCATATCAGGATTATCAATACCATATTTTGAAAAAAGCCGTTTCTGTAATGAAG GAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCAAAATCACTC GCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCTGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATA AAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAACATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGGTAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATAATAAATCAGCATCCATGTTGGAATTTAATCGC GGCCTTGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAA AGCAAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCATCAACCGTGGCTCCCTCACTTTCTGGCTGGATGATGGGCGATTCAGGCGATCCCCATCCAACAGCCCGCCGTCGAGCGGGCT.

In the above-mentioned embodiments, the descriptions of each embodiment have their own emphases, and for parts that are not detailed or recorded in a certain embodiment, refer to the relevant descriptions of other embodiments.

2. Application examples:

Application example

The application example of the present invention provides a barley HvFRF1 gene, the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO.1. The barley HvFRF1 gene is a gene cloned from barley.

The application example of the present invention also provides a protein encoded by the barley HvFRF1 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

The application example of the present invention also provides an Arabidopsis overexpression vector super1300:HvFRF1 constructed by homologous recombination based on the barley HvFRF1 gene.

The application example of the present invention also provides a barley overexpression vector pBract214:HvFRF1 constructed by the Gateway method based on the barley HvFRF1 gene.

The application example of the present invention also provides an application of the barley HvFRF1 gene in obtaining a drought-resistant Arabidopsis strain.

The application example of the present invention also provides an application of the barley HvFRF1 gene in obtaining drought-resistant barley lines.

The application example of the present invention also provides an application of barley HvFRF1 gene in tissue expression in barley root system induced by drought stress.

The above is only a preferred specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technical field is within the technical scope disclosed in the present invention Any modifications, equivalent replacements and improvements made within the spirit and principles shall fall within the protection scope of the present invention.

Claims (10)

1. A barley HvFRF1 gene is characterized in that the nucleotide sequence of the barley HvFRF1 gene is shown in SEQ ID NO. 1.

2. The barley HvFRF1 gene according to claim 1, characterized in that the barley HvFRF1 gene is a gene cloned from barley.

3. The barley HvFRF1 gene according to any one of claims 1 to 2, which encodes a protein having the amino acid sequence shown in SEQ ID No. 2.

4. An Arabidopsis thaliana overexpression vector super1300: hvFRF1 constructed by homologous recombination according to the barley HvFRF1 gene of claim 1, the nucleotide sequence of which is shown as SEQ ID No. 26.

5. A barley overexpression vector pBract214 HvFRF1 constructed by the Gateway method according to the barley HvFRF1 gene of claim 1, the nucleotide sequence of which is shown as SEQ ID NO. 29.

6. An application of barley HvFRF1 gene in obtaining drought-resistant arabidopsis thaliana strains.

7. An application of a barley HvFRF1 gene in obtaining a barley strain with drought resistance.

8. An application of barley HvFRF1 gene in tissue expression in barley root system under the induction of drought stress.

9. An application of barley HvFRF1 gene in controlling arabidopsis thaliana to screening arabidopsis thaliana varieties.

10. An application of a barley HvFRF1 gene in screening barley varieties by regulating and controlling the drought resistance of barley.

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