CN108588088A - A kind of drought resisting transcription factor PbrERF109 and preparation method thereof, application and coding protein and application - Google Patents

Abstract

The present invention provides a kind of drought resisting transcription factor PbrERF109 and preparation method, the protein of application and coding and applications, belong to field of plant genetic, have nucleotide sequence shown in ESQ ID No.1.Drought resisting transcription factor PbrERF109 is transferred in tobacco and Ussurian pear by the present invention, obtained transgenosis overexpression strain drought-resistant ability compared with compareing wild type has very big promotion, the content of hydrogen peroxide and malonaldehyde is intended to lower than wild type in the transgenosis overexpression strain of tobacco, plant activity in vivo oxygen residual is lower, cellular damage smaller, and then improve the drought-resistant ability of tobacco and Ussurian pear plant.

Description

A drought-resistant transcription factor PbrERF109 and its preparation method, application and encoded protein White matter and application

technical field

The invention belongs to the field of plant genetic engineering, and specifically relates to a drought-resistant transcription factor PbrERF109, its preparation method, application, encoded protein and application.

Background technique

Plants often encounter some adversity stresses during their growth cycle, including biotic stresses and abiotic stresses. Abiotic stresses mainly include drought, high temperature, low temperature, heavy metal stress, etc. Among them, drought affects the growth and development of plants, Yield quality and one of the important factors limiting the geographical distribution of plants. It is now clear that the survival of plants under drought stress mainly depends on the metabolism in the body and the adaptive changes in plant morphology, including a series of physiological and biochemical reactions to establish a systemic defense state and respond to drought stress at multiple levels , the expression of many drought stress-responsive genes is regulated during this adaptation process (Pastori and Foyer, 2002). Great strides have been made in identifying signal transduction elements involved in stress-responsive responses.

So far, many key genes involved in enhancing plant stress resistance or tolerance have been verified, and these are generally classified into two types. One is composed of functional proteins that directly function to prevent cells from being damaged by stress, and the other is composed of regulatory proteins that regulate signal transduction and gene expression processes (Chaves et al., 2003; Shinozaki et al., 2003; Yamaguchi -Shinozaki and Shinozaki, 2005; Bartels and Sunkar, 2005). In the process of people studying adversity stress, transgenic plants with enhanced resistance through transgenesis are excellent research materials (Ward and Schroeder, 1994; Klein et al., 2004; Shinozaki and Yamaguchi-Shinozaki, 2007). Studies have shown that the stress resistance of plants can be significantly improved by overexpressing regulatory factors and functional genes. However, the results of constitutive expression of functional genes and transcription factors will be different (Agarwal et al., 2006), and the role of transcription factors in improving plant stress resistance is better than that of functional genes. A transcription factor can regulate the expression levels of a series of downstream genes, thereby jointly exerting stress resistance in various abiotic stresses (including drought) (Century et al., 2008; Yang et al., 2011). Therefore, genetic transformation of transcription factors is an important way and means for genetic improvement of plant resistance.

There are many transcription factors in the plant genome, and ERF (Ethylene responsive factors, ethylene response factors) transcription factors are indispensable and important members of the regulatory network. This type of transcription factor family protein has an AP2/ERF domain, which is It consists of an α-helix and three antiparallel β-sheet structures. The α-helix interacts with the DNA major groove through the hydrophobic side to bind to DNA, while the β-sheet functions to recognize the promoter element of the target gene, in which the 14th and 19th amino acid residues of the second β-sheet Determines the binding body specificity of ERF transcription factors (Okamuro et al., 1997). ERF transcription factors are ubiquitous in plants, for example, there are 147 and 157 genes in Arabidopsis and rice, respectively (Jofuku et al., 1994; Nakano et al., 2006). Since ERF transcription factors only exist in plants, their research is relatively extensive. It has been reported that ERF transcription factors have certain effects on the growth and development of plant organs such as seeds, roots, leaves and flowers (Xie et al., 2017). Some ERF proteins, for example, the pathogen-induced ERF1 gene (TaPIE1) in wheat responds to ethylene signaling and activates downstream defense and stress-related genes, resistance to plant necrotrophic Rhizoctonia cerealis infection and cold stress It has played a positive regulatory role (Zhu et al., 2014). Ethylene and ABA signals can induce the GhERF4 gene in cotton and play a key role in the response of cotton to abiotic stress (Jin and Liu, 2008). However, there is no report in the prior art that ERF transcription factors can resist drought.

Contents of the invention

In view of this, the object of the present invention is to provide a drought-resistant transcription factor PbrERF109, which can improve the drought-resistant ability of plants.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a drought-resistant transcription factor PbrERF109, which has the nucleotide sequence shown in ESQ ID No.1.

The present invention also provides that the protein encoded by the drought-resistant transcription factor PbrERF109 described in the technical solution has the amino acid sequence shown in SEQ ID No.2.

The present invention also provides a method for preparing the drought-resistant transcription factor PbrERF109 described in the above technical scheme, comprising the following steps:

Using pear cDNA as a template, the drought-resistant transcription factor PbrERF109 was obtained by PCR amplification with transcription factor primers;

The transcription factor primer pair includes a transcription factor upstream primer and a transcription factor downstream primer, the transcription factor upstream primer has the nucleotide sequence shown in SEQ ID No.3, and the transcription factor downstream primer has the nucleotide sequence shown in SEQ ID No.4 The nucleotide sequence shown.

Preferably, each 50 μl of the PCR amplification system includes: 100 ng pear cDNA, 10 μl Q5ReactionBuffer, 1 μl 10 mM dNTP, 0.5 μl Taq polymerase, 2.5 μl 10 μM transcription factor upstream primer, 2.5 μl 10 μM transcription factor downstream primer, and the balance is ddH ₂ O;

The PCR amplification conditions include: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; and extension at 72°C for 2 minutes after the cycle is completed.

The present invention also provides the application of the drought-resistant transcription factor PbrERF109 described in the above technical scheme or the protein in improving the drought-resistant ability of plants.

Preferably, said plant comprises tobacco or pear.

Preferably, when the plant is tobacco, the method for improving the drought resistance of tobacco comprises the following steps:

connecting the drought-resistant transcription factor PbrERF109 with the pEASY-T1 cloning vector to obtain a recombinant vector;

Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

The recombinant Agrobacterium was used to infect tobacco.

Preferably, each 5 μl of the total system connected with the pEASY-T1 cloning vector includes: 4 μl of drought-resistant transcription factor PbrERF109 and 1 μl of the EASY-T1 cloning vector, and the molar ratio of the drought-resistant transcription factor PbrERF109 to the pEASY-T1 cloning vector is 3:1;

The connection conditions include: reacting at 25° C. for 30 minutes.

Preferably, when the plant is pear, the method for improving the drought resistance of pear comprises: transferring the drought-resistant transcription factor PbrERF109 into pear by using a transient transformation method.

The expression of the drought-resistant transcription factor PbrERF109 provided by the present invention can effectively enhance the active oxygen scavenging ability of transgenic plants, thereby improving the drought-resistant ability of the plants.

According to the results of the examples of the present invention, the present invention transfers the drought-resistant transcription factor PbrERF109 into tobacco and Qiuzi pear. The content of hydrogen peroxide and malondialdehyde in the expression line is lower than that of the wild type, the residual reactive oxygen species in the plant is lower, and the cell damage is smaller, thereby improving the drought resistance of tobacco and Qiuzi pear plants.

Description of drawings

Fig. 1 is a technical flow chart of the present invention;

Fig. 2 is the expression of the PbrERF109 gene of the present invention under dehydration, abscisic acid and 4°C low temperature stress;

Figure 3 is the subcellular localization of the PbrERF109 gene of the present invention, wherein: Figure 3-A, the imaging of the GFP gene (control) in bright field (in the figure) and ultraviolet light (left in the figure), and the figure (right) is the superimposition of the two After imaging; Fig. 3-B, the imaging of PbrERF109 gene under bright field (middle) and UV light (left), and the image (right) is the superimposed image of the two;

Figure 4 is the PbrERF109 gene transcription activation identification of the present invention, Figure 4 pGBKT7 ^TM is the growth of yeast transformed with empty vectors on different media; pGBKT7 ^TM -PbrERF109 is the growth of yeast transformed with fusion vectors on different media;

Fig. 5 is pCAMBIA1301 plasmid map;

Fig. 6 is the flow chart of constructing the plant overexpression vector of PbrERF109 gene;

Fig. 7 is a schematic diagram of PbrERF109 transformed tobacco and plant regeneration process;

Figure 8 is a schematic diagram of PCR identification of PbrERF109 gene transgenic plants, Figure 8-A uses PbrERF109 gene-specific primers for PCR identification _of T1 generation transgenic plants after tobacco, M: Maker, P: plasmid pCAMBIA1301-PbrERF109, WT: wild type Plants, 1, 2, 3, ..., 7, 8: transgenic lines; Figure 8-B is the expression level analysis of exogenous gene PbrERF109 in tobacco transgenic plants, WT is wild type, and others are transgenic lines;

Fig. 9 is the determination of phenotype and physiological index before and after drought treatment of PbrERF109 gene lines (OE5 and OE8) and wild type (WT) non-transgenic plants (WT) in the embodiment of the present invention, wherein Fig. 9-A is 30 days old The phenotypes of tobacco plants in normal watering for 3 days and drought treatment for 20 days; Figure 9B-E is the survival rate, water loss rate and electrical conductivity of 30-day-old tobacco plants in normal watering for 3 days and drought treatment for 20 days And chlorophyll content statistics;

Figure 10 is the histochemical staining analysis of H ₂ O ₂ and O ² -accumulation and malondialdehyde of PbrERF109 gene-transferred lines (OE5 and OE8) and wild-type (WT) non-transgenic plants (WT) in the examples of the present invention after drought treatment Figure 10, AB is a 30-day-old tobacco plant after 3 days of normal watering and 20 days of drought treatment, non-transformed plants and two transgenic lines Histochemical staining of reactive oxygen species, using diaminobenzidine and Nitrotetrazolium stained H ₂ O ₂ ( FIG. 10-A ) and O ^2- ( FIG. 10-B ), respectively; FIG. 10-C : staining diagram of cell death after drought stress treatment of transgenic tobacco. Fig. 10D is a graph showing the determination of malondialdehyde content in transgenic tobacco after drought stress treatment.

Detailed ways

The invention provides a drought-resistant transcription factor PbrERF109, which has the nucleotide sequence shown in ESQ ID No.1. In the present invention, the drought-resistant transcription factor PbrERF109 can improve the drought-resistant ability of plants. In the present invention, the full length of the drought-resistant transcription factor PbrERF109 is 795 bp, including an open reading frame of 795 bp, encoding 264 amino acids, an isoelectric point of 5.99, and a molecular weight of 28.9 KDa.

In the present invention, the drought-resistant transcription factor PbrERF109 has the nucleotide sequence shown in ESQ ID No.1, and the specific sequence is as follows:

atgcccttccatgcgaatcggatacaacaggagcaggagcactgcatcatggtctccgccctcaagcacgtaatctccggtggaagcatcagtgggcccacacctcagccaatgccggcggtctacaatgccacgtcatccgtctcgacgagcggcacccagttggcagcgggccaaccagcacaacaggacaactacttctcgccatcgttgccgaatcaaaacaggaaccagcaactgagtttgggaaccgggtttgtcgggatgaatgcgccaactacgaggaagagcaagaacaagtacaggggcgtcaggcagaggccgtgggggaaatgggcggcggagattcgagacccacgacgggcggcgagggtgtggctagggacgttcgagacggcggaggacgcggccagggcttacgacaaggccgccgtcgagttccgcggaaataaggcaaagctcaatttcccatcggacccgggcggtcacattgtcacgactaacgacagttctagtagtggaactagtgctaatgccagtattaatccaggattaattaataagcaaaagcaaaagaatattagcgaaattggggtcatggagaaggaggaggagaaggttgatcaggtcaaactcaaccaggcgacgcagccggagaatatgcaactgggggtggtggcgaccgcggagagcagcgttggtcatgaggaggatgaccagttcttgttgtgggacaatggctggctccgagatggtgaagatgacgacttaatggcatggttatccacgaactag。

In the present invention, the preparation method of the drought-resistant transcription factor PbrERF109 comprises the following steps:

Using pear cDNA as a template, the drought-resistant transcription factor PbrERF109 was obtained by PCR amplification with transcription factor primers;

The transcription factor primer pair includes a transcription factor upstream primer and a transcription factor downstream primer, the transcription factor upstream primer has the nucleotide sequence shown in SEQ ID No.3, and the transcription factor downstream primer has the nucleotide sequence shown in SEQ ID No.4 The nucleotide sequence shown.

In the present invention, the method for preparing pear cDNA preferably comprises: extracting RNA from leaves of Pear pear, and reverse-transcribing the obtained RNA to obtain cDNA. The method for extracting RNA is not particularly limited in the present invention, and the method for extracting plant RNA conventionally used by those skilled in the art can be used.

In the present invention, the transcription factor PbrERF109 is preferably derived from Pyrusbretschneideri, and the primer pair is designed according to the open reading frame of the gene PbrERF109. In the present invention, the upstream primer of the transcription factor has the nucleotide sequence shown in SEQ ID No.3, specifically as follows:

5'-GA AGATCT TATGCCCTTCCATGCGAATCGGATA-3';

The transcription factor downstream primer has the nucleotide sequence shown in SEQ ID No.4, specifically as follows:

5'-GGGTNACCCTAGTTCGTGGATAACCA-3'.

In the present invention, each 50 μl of the PCR amplification system preferably includes: 100 ng pear cDNA, 10 μl Q5 Reaction Buffer, 1 μl 10 mM dNTP, 0.5 μl Taq polymerase, 2.5 μl 10 μM transcription factor upstream primer, 2.5 μl 10 μM transcription factor downstream primer , the balance being ddH ₂ O. In the present invention, the Q5 Reaction Buffer and Taq polymerase are preferably purchased from NEB Company, which is produced in the United States.

In the present invention, the enzyme activity of the Taq polymerase is preferably 1U.

In the present invention, the PCR amplification conditions preferably include: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; extension at 72°C after the cycle is completed 2 minutes.

The present invention also provides the application of the drought-resistant transcription factor PbrERF109 obtained in the above-mentioned technical solution or the protein in improving the drought-resistant ability of plants. In the present invention, the specific way to improve the drought resistance ability of plants is to construct transgenic plants, and transfer the drought resistance transcription factor PbrERF109 into plants.

In the present invention, the plant preferably includes tobacco or pear, and the pear is preferably Qiuzi pear.

In the present invention, when the plant is tobacco, the method for improving the drought resistance of tobacco is to construct transgenic tobacco, which specifically includes the following steps:

connecting the drought-resistant transcription factor PbrERF109 with the pEASY-T1 cloning vector to obtain a recombinant vector;

Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

The recombinant Agrobacterium was used to infect tobacco.

In the present invention, the total system connected with the pEASY-T1 cloning vector preferably includes every 5 μl: 4 μl of drought-resistant transcription factor PbrERF109 and 1 μl of the EASY-T1 cloning vector, and the molar ratio of the drought-resistant transcription factor PbrERF109 to the pEASY-T1 cloning vector is preferably 3:1.

In the present invention, the connection conditions preferably include: reacting at 25° C. for 30 minutes.

The present invention has no special limitation on the type of the vector, and those conventionally selected by those skilled in the art can be used. In the embodiment of the present invention, the vector is pEASY-T1.

In the present invention, there is no special limitation on the method for transferring the recombinant vector into Agrobacterium tumefaciens to obtain the recombinant Agrobacterium tumefaciens, and the conventional method of transferring the vector into Agrobacterium tumefaciens can be adopted by those skilled in the art.

In the present invention, the method for infecting tobacco with the recombinant Agrobacterium is not particularly limited, and the conventional method for infecting tobacco with the recombinant Agrobacterium can be adopted by those skilled in the art.

In the present invention, when the plant is pear, the method for improving the drought resistance of pear comprises: transferring the drought-resistant transcription factor PbrERF109 into pear by using a transient transformation method.

The present invention has no special limitation on the transient transformation method adopted, and the conventional transient transformation method selected by those skilled in the art can be used.

The present invention also provides that the protein encoded by the drought-resistant transcription factor PbrERF109 described in the above technical scheme has the amino acid sequence shown in SEQ ID No.2, and the specific sequence is as follows:

Met Pro Phe His Ala Asn Arg Ile Gln Gln Glu Gln Glu His Cys Ile MetVal Ser Ala Leu Lys His Val Ile Ser Gly Gly Ser Ile Ser Gly Pro Thr Pro GlnPro Met Pro Ala Val Tyr Asn Ala Thr Ser Ser Val Ser Thr Ser Gly Thr Gln LeuAla Ala Gly Gly Gln Pro Ala Gln GlnAsp Asn Tyr Phe Ser Pro Ser Leu Pro Asn GlnAsn Arg Asn Gln Gln Leu Ser Leu Gly Thr Gly Phe Val Gly Met Asn Ala Pro ThrThr Arg Lys Ser Lys Asn Lys Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys TrpAla Ala Glu Ile Arg Asp Pro Arg Arg Ala Ala Arg Val Trp Leu Gly Thr Phe GluThr Ala Glu Asp Ala Ala Arg Ala Tyr Asp Lys AlaAla Val Glu Phe Arg Gly AsnLys Ala Lys Leu Asn Phe Pro Ser Asp Pro Gly Gly His Ile Val Thr Thr Asn AspSer Ser Ser Ser Gly Thr Ser Ala Asn Ala Ser Ile Asn Pro Gly Leu Ile Asn LysGln Lys Gln Lys Asn Ile Ser Glu Ile Gly Val Met Glu Lys Glu Glu Glu Lys ValAsp Gln Val Lys Leu Asn Gln Ala Thr Gln Pro Glu Asn Met Gln Leu Gly ValValAla Thr Ala Glu Ser Ser Ser Val Gly His Glu Glu Asp Gln Phe Leu Leu TrpAsp Asn Gly Trp Leu Arg Asp Gly Glu Asp Asp Asp Leu Met Ala Trp Leu Ser Thr Asn.

A drought-resistant transcription factor PbrERF109 provided by the present invention and its preparation method, application, encoded protein and application will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

Cloning and expression analysis of PbrERF109 gene

RNA was extracted from the leaves of Duli pear under drought and dehydration treatment, and cDNA was obtained by reverse transcription. In the present invention, after obtaining the cNDA, the obtained cDNA is used as a template, and a specific transcription factor primer pair is used to obtain the drought-resistant transcription factor PbrERF109, the specific sequence of which is shown in SEQ ID No.1.

For RNA extraction, Plant Total RNA Isolation Kit Plus (Foregene, RE-05022) was used, and the operation instructions provided by the kit were followed. First-strand cDNA was synthesized using First Script Strand cDNASynthesis SuperMix (Transgene, AE301-02) reverse transcription kit (operated according to the instructions provided by the kit).

The primer pair for amplifying the drought-resistant transcription factor PbrERF109 is: forward primer: PbrERF109Forward, 5'-GA AGATCT TATGCCCTTCCATGCGAATCGGATA-3' (SEQ ID No.3); reverse primer: PbrERF109Reverse, 5'-G GGTNACC CCTAGTTCG TGGATAACCA-3 ' (SEQ ID No. 4).

The 50 μl reaction system included 100 ng cDNA, 10 μl 5× buffer (Q5Reaction Buffer), 1 μl 10 mM dNTP, 0.5 μl 1U Taq polymerase (Q5 High-Fidelity DNA Polymerase) (the aforementioned buffer and Taq polymerase were purchased from NEB Company, USA ), each 2.5 μl of 10 μM forward and reverse primers, the enzymatic activity of Taq polymerase is 1U. The PCR reaction was completed on the Veriti Thermal Cycler (Applide Biosystem) amplification instrument according to the following procedures: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; cycle completion Then extend at 72°C for 2 minutes. Produce a single PCR band product, after 1% agarose gel electrophoresis, use DNA gel extraction kit (purchased from Axygen, USA) was used to recover specific bands, and the extraction steps refer to the instructions for use.

The recovered and purified DNA solution was ligated with the pEASY-T1 carrier (purchased from Quanshijin Company) and operated according to the instructions. The molar ratio of the inserted PbrERF109 gene and the pEASY-T1 carrier in the ligated reaction system was 3:1. The total volume of the ligated reaction was 5 μl, including 4 μl of purified PCR product, 1 μl of T vector, ligated at 25°C for 30 minutes.

Take 5 μl of the ligation product, transform Escherichia coli DH5α by heat shock method (referring to the third edition of "Molecular Cloning Experimental Manual", Science Press, 2002), and screen positive clones on LB solid plates containing 50 mg/L kanamycin, Pick 8 clones for sequencing (completed by Nanjing Yidao Biotechnology Co., Ltd.), and the sequencing results show that the cloned PbrERF109 gene of the present invention has a full length of 795bp, and it is determined by sequencing and comparison that it is the target gene required by the present invention. The sequence is as SEQ ID No .1, the gene was named PbrERF109, which contains a coding reading frame of 795 bp, encodes 264 amino acids, has an isoelectric point of 5.99, and a predicted molecular weight of 28.9 KDa. BLASTX analysis of this sequence is homologous to the known sequence of ERF1 09 (all published literature and databases). Although the amino acid sequence of the deduced PbrERF109 gene has 85% homology with the predicted sequence of apple (MdERF109, XP_008338309), the function of apple MdERF109 gene is unknown. Multiple sequence alignment results showed that PbrERF109 has a conserved domain, AP2/ERF domain. SMART predicts amino acid PbrERF109 to have 1 transcriptional activation region.

To analyze whether the PbrERF109 gene responds to drought, abscisic acid (ABA), and low temperature treatments, real-time fluorescent quantitative PCR (forward primer PbrERF109-F1: 5'-AGCCGGAGAATATGCAACTG-3' (SEQ ID No.5); reverse Primer PbrERF109-R1: 5'-TGTCCCACAACAAGAACTGG-3' (SEQ ID No. 6)) was used to observe the expression of the gene under different stress treatments, so as to determine which stress induced the gene PbrERF109 most strongly. The RNA was extracted by the CTAB method, and the synthesis of the first strand of cDNA was carried out according to the operation manual of the TOYOBO reverse transcription kit. In the 20 μl reaction system, there are: 10 μl 2×Mix, 0.1 μl cDNA, 5 μl primer (with Tublin as the internal reference primer, the length is 208), 4.9 μl water. The procedure of quantitative PCR is as follows:

Table 1 Quantitative PCR program

When the plants were subjected to dehydration treatment, as shown in Figure 2A, the transcription level of the PbrERF109 gene gradually increased after the plants were dehydrated, and reached the highest level at 0.5h, which was nearly 4 times that of untreated plants, indicating that the PbrERF109 Genes respond strongly to dehydration. When the plants were treated with abscisic acid, as shown in Figure 2B, the transcription level of the PbrERF109 gene gradually increased after the plants were treated with abscisic acid, and reached the highest level at 1 h, which was more than three times that of untreated plants, indicating that The PbrERF109 gene is strongly responsive to abscisic acid. When the plants were treated with low temperature at 4°C, as shown in Figure 2C, the transcription level of PbrERF109 gene increased slowly at 6h, then gradually decreased to 72h, and finally increased gradually at 144h. There was no obvious change from 6h to 72h, which was more than 1 times that of untreated, indicating that the response of PbrERF109 gene to 4°C low temperature was not obvious. The results showed that drought-dehydration treatment could induce the expression of this gene, indicating that it was a drought-responsive candidate gene.

Example 2

Analysis of subcellular localization and transcriptional activation of PbrERF109 gene

Since the PbrERF109 gene is a transcription factor, the present invention uses tobacco mesophyll cells to study the subcellular localization of the PbrERF109 gene. The entire ORF reading frame of the PbrERF109 gene was amplified by RT-PCR, and two restriction sites, XbaI and BamHI, were added at both ends of the amplification primer. The amplification primers are (forward primer PbrERF109-F2: 5'- TCTAGA ATGCCCTTCCATGCGAATCGGATA-3' (SEQ ID No.11); reverse primer PbrERF109-R2: 5'- GGATCC CCTAGTTCGTGGATAACCA-3' (SEQ ID No.12 )), firstly digest the amplified product. In the present invention, the temperature for the double digestion of the pear drought-induced transcription factor PbrERF109 is 37° C., and the time for the double digestion is 12 hours; the total volume of the double digestion system is 20 μl, including the PCR purified product of the pear drought-induced transcription factor PbrERF109 10 μl, 2 μl of 10×G buffer, 1 μl of XbaI and BamHI, 6 μl of double distilled water. At the same time, pCAMBIA1302 was digested with XbaI and BamHI, and the product was recovered and ligated to obtain the pCAMBIA1302-PbrERF109-GFP recombinant vector, which was transformed into Agrobacterium EHA105. Agrobacterium infection of tobacco mesophyll cells is carried out as follows: (1) pick the Agrobacterium monoclonal on the fresh medium and inoculate it in 5ml LB/Kan/Rif liquid medium (containing 50mg/L kanamycin and 50mg/L lizard Fuping), cultured at 28°C for 1-2 days, 220rpm/min, activated bacterial liquid; (2) Take activated bacterial liquid and inoculate it into 50ml LB/Kan/Rif liquid medium (containing 50mg/L Kan Mycin and 50mg/L rifampicin), cultured at 28°C for 8-12 hours, 220rpm/min, during the cultivation period, the OD600 of the bacterial liquid was detected to be between 0.6 and 0.8; (3) Transfer the bacterial liquid to a 50ml centrifuge tube, 4000rpm/min, centrifuge for 5 minutes, remove the supernatant; (4) Add 10ml cleaning solution (10mM MES+10mM MgCl2) to resuspend the bacteria, 4000rpm/min, centrifuge for 5 minutes, remove the supernatant, and repeat with 5ml cleaning solution , finally add 3ml cleaning solution, fully suck and mix; (5) draw resuspended bacteria liquid, add in cleaning solution at 19:1, measure OD ₆₀₀ ; (6) injection is 5ml/combination, adjust two kinds of bacterial solutions The final concentration OD ₆₀₀ is about 0.6, and finally make up to 5ml with cleaning solution, add 5μl acetosyringone (AS), mix well, place at room temperature for 2-3 hours, and wait for injection; (7) Put the infection solution into In the syringe (5ml), inject 2-3 leaves in one combination. The leaves are randomly selected and distributed on different plants, and the leaves of moderate size are picked up, and the liquid is injected into the lower epidermis of the tobacco leaves by pressing the syringe; (8) After the injection, Place the tobacco at 25°C for 48-72 hours; (9) inject 4',6-diamidino-2-phenylindole into the leaves of the two for nuclear staining, and then use a confocal laser microscope ( Zeiss LSM 710, Germany) observed the localization of the reporter gene, and the results showed that there was fluorescence in the whole cell when the control vector was transformed, as shown in Figure 3A, while the fluorescence in the cells transformed with the recombinant vector could only be detected in the stained area of the nucleus specific dye DAPI The results are shown in Figure 3B, indicating that PbrERF109 is a nuclear localized protein.

Example 3

Plant Transformation Overexpression Vector Construction

According to the multiple cloning site of the pCAMBIA1301 vector ( FIG. 5 ) and the restriction site analysis on the coding region sequence of the PbrERF109 gene, BglII and BsteII were selected as endonucleases. First, the clone of the PbrE RF109 gene was used as a template for PCR amplification (amplification primer pair forward primer: PbrERF109Forward, 5'-GA AGATCT TATGCCCTTCCATGCGAATCGGATA-3' (SEQ ID No.3); reverse primer: PbrERF109Reverse, 5''-G GGTNACC CCTAGTTCGTGGATAACCA-3' (SEQ ID No.4)), and then digest the PCR product together with the pCAMBIA1301 vector at 37°C for 2-3 hours and then purify and recover. The ratio of the amount of the PbrERF109 gene to the vector pCAMBIA1301 was 3:1 in the ligation reaction system, and the total reaction volume was 10 μl. It contains: 1 μl of 10×buffer, 1 μl of T4 DNA ligase, 4ul of PbrWRKY53 gene recovered by double enzyme digestion, 2μl of pCAMBIA1301 vector product recovered by double enzyme digestion, 2μl of double distilled water, and react at 16°C for 14-16h to obtain the ligation product. The ligation product was transformed into Escherichia coli strain DH5α, screened on LB solid plate containing 50mg/L kanamycin, and the single clone was selected to be positive by PCR, and the plasmid was extracted for enzyme digestion. After the sequence was confirmed to be correct, pCAMBIA1301-PbrERF109 recombination Vector construction was successful. The recombinant vector was introduced into Agrobacterium tumefaciens GV3101 by freeze-thaw method and kept. The construction process of the plant overexpression vector 'pCAMBIA1301-PbrERF109' is shown in Figure 6.

Example 4

tobacco genetic transformation

The genetic transformation steps of tobacco mediated by Agrobacterium tumefaciens are as follows:

1. Agrobacterium culture: Take the freshly activated Agrobacterium liquid, mark it on the LB solid plate with 50mg/L kanamycin and 50mg/L rifampicin, and scrape it with a sterilized pipette after 2 days Put the colony on the plate into the liquid MS, 28C °, 200 rpm shaking culture, and stop the culture when the concentration of the bacterial solution reaches OD ₆₀₀ = 0.4 ~ 0.6, and then use it for dipping;

2. Dip dyeing: Take healthy non-transgenic wild-type tobacco leaves, cut them into squares with a size of about 0.5 cm in length and width, put them into the cultured Agrobacterium GV3101 liquid with tweezers, submerge and soak for about 10 minutes, During the cultivation period, place on a shaker at 28°C and 200rpm and shake continuously;

3. Co-cultivation: Gently take out the leaves dipped in MS with tweezers, blot the excess bacterial solution on the surface with sterile filter paper, and then clamp the leaves (leaf surface facing up) and place them on the symbiotic medium. Dark treatment at 25°C for 72 hours of cultivation;

4. Screening culture: Take out the leaves that have been cultured in the dark, put the leaves into the prepared cephalosporin solution, soak them, rinse once, then rinse the leaves with sterile water for 4 times, and dry the surface residue with sterile filter paper Moisture, finally put blade (leaf surface down) in the MS medium containing 20mg/L hygromycin and 500mg/L cephalosporin;

5. Rooting culture: Tobacco leaves will grow adventitious buds on the screening medium. When the adventitious buds are estimated to be about 1 cm long by visual inspection, cut off the adventitious buds, and carefully put them in with tweezers containing 20mg/L hygromycin, 500mg/L cephalosporin and 0.5mg/L 6-BA MS medium;

6. Tobacco seedling transplanting: When observing the growth of the transgenic tobacco seedlings to a certain extent, clamp the stem with tweezers, pull it out from the rooting medium, rinse the MS medium at the root of the transgenic tobacco seedlings with water, and place in The aseptic nutrient soil mixed with vermiculite and clean pots were transplanted to obtain transgenic tobacco plants of PbrERF109.

Example 5

Instant transformation of pears

The preparation of Qiuzi pear strain transiently transformed with pear drought-induced transcription factor PbrERF109, the specific method is as follows:

1. Select Pyrus ussuriensis grown in an artificial climate chamber for about 5 weeks for Agrobacterium infection.

2. In LB medium (containing 50mg/L kanamycin + 100mg/L rifampicin + 50mg/L gentamycin) streak culture GV3101 Agrobacterium with the target plasmid, pick the Agrobacterium clone and culture it overnight at 28°C in 5mL LB medium .

3. After measuring the OD600 value of the Agrobacterium bacterial liquid, centrifuge at 3000 rpm for 10 minutes to collect the bacterial liquid, and discard the supernatant. Use acetosyringone (acetosyringone) solution [10mM MES (pH 5.6) + 10mM MgCl2 + 200uM acetosyringone] to suspend, adjust to OD600 of about 1.5, and let stand at room temperature for 3h.

4. The Agrobacterium containing the objective plasmid was used to inject the leaves of Qiuzi pear.

5. Use a 1mL syringe (remove the needle), inject on the back of the pear leaves, and mark the injected leaves.

6. Put the injected pear plants back into the artificial climate chamber for 6-7 days to observe the results of instantaneous transformation.

Example 6

Molecular identification of transgenic plants

1. Leaf DNA extraction

Take an appropriate amount of transgenic tobacco plant leaves and put them into a 1.5ml centrifuge tube, add liquid nitrogen, and grind them thoroughly until they are powdered; immediately add 700 μl 65°C preheated DNA extraction solution cetyl triethyl to the centrifuge tube that has been ground into powder ammonium bromide, extract formula: 100mM Tris-HCl (PH8.0), 1.5M NaCl, 50mM EDTA (PH=8.0), 1% polyvinylpyrrolidone, 2% CTAB; Put it in a 65°C water bath for 90 minutes, take it out and mix it upside down every 15 minutes; after warming, centrifuge at 10000g at room temperature for 10 minutes, take the supernatant, add 600μl chloroform, mix it upside down, and let it stand at room temperature for 5 minutes; centrifuge at 10000g for 15 minutes Minutes, take the supernatant, 2 times the volume of pre-cooled absolute ethanol, 34μl 5M NaCl solution, mix it upside down, put it in a -20℃ refrigerator for 30 minutes; centrifuge at 10000g for 10 minutes, take the supernatant, and use 1ml Wash the precipitate with % ethanol for 3 times, centrifuge the empty tube for one minute, and dry the alcohol until the DNA is colorless and transparent. Gel electrophoresis detection.

2. Semi-quantitative RT-PCR detection of overexpression of PbrERF109 gene

In this study, semi-quantitative RT-PCR was used to analyze the expression level of exogenous gene PbrERF109 in transgenic pear and tobacco plants, and the methods of RNA extraction and cDNA synthesis from leaves of transgenic lines were the same as in Example 1. The primers used for semi-quantitative analysis were PbrERF109 gene-specific primers (forward primer PbrERF109-F1: 5'-AGCCGGAGAATATGCAACTG-3' (SEQ ID No.5); reverse primer PbrERF109-R1: 5'-TGTCCCACAACAAGAACTGG-3' (SEQ ID No. .6)). The reaction program was pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and extension at 72°C for 5 minutes after the cycle was completed. The expression of exogenous genes in transgenic tobacco was analyzed by semi-quantitative PCR, and Ubiqutin was used as an internal reference for the analysis of transgenic tobacco. The primer sequence was: forward primer Ubiqutin-F: 5'-TGGCGGACGGGTGAGTAACGCG-3' (SEQ ID No.7); reverse primer Ubiqutin -R: 5'-GCTGGTCCGGTGGGATACCCTCC-3' (SEQ ID No. 8). Transgenic pear analysis uses Tublin as an internal reference, forward primer Tublin-F: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID No.9); reverse primer Tublin-R: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID No.10) . The results showed that the expression levels of the PbrERF109 gene in the transgenic lines were higher than those of the wild type, and 5 and 8 with high brightness were selected, that is, the two overexpression lines with high expression levels were named OE5 and OE8 as separate transgenic lines, Then they were used as female plants for harvesting seeds respectively.

Example 7

Resistance Evaluation of Transgenic Plants

Tobacco untransformed plants (WT) and PbrERF109 overexpression lines (OE5, OE8) received in the same batch were transplanted into pots after growing WT, OE5 and OE8 on MS growth medium for 15 days and growing for 20 days, and the normal growing plants There was no difference, but after 20 days of drought treatment, the WT plants wilted severely, while the OE5 and OE8 plants still grew normally, indicating that the drought resistance of the transgenic plants was significantly stronger than that of the wild type (Fig. 9A). The survival rate was counted, and the results showed that the survival rate of the two transgenic lines was significantly higher than that of the wild type ( FIG. 9B ). Figure 9 (C, D) respectively show the water loss rate and electrical conductivity of the plants after the drought treatment, and the results show that the water loss rate and electrical conductivity of the transgenic plants are significantly lower than those of the wild type. Figure 9E is the measurement of chlorophyll and the extraction of chlorophyll after 20 days of drought treatment at room temperature. The above studies showed that the drought resistance ability of tobacco plants transfected with PbrERF109 gene was significantly enhanced compared with non-transformed plants (WT).

In the transgenic lines, the lower conductivity and higher survival rate indicated that they may have stronger ROS resistance than WT. Then it becomes necessary to identify the accumulation of ROS in plants. The leaves of the plants were stained with DAB and NBT histochemical staining methods to detect the content of hydrogen peroxide (H ₂ O ₂ ) and superoxide anion (O ^2- ), respectively. As shown in Figure 10 (A, B) after 20 days of drought stress, the leaves stained with DAB, the brown leaf area of the wild-type strain was significantly larger than that of the transgenic strain, and the color was darker, and the leaves stained with NBT, the wild-type strain The blue color of the line is darker and larger than that of the transgenic line. Similarly, as shown in Figure 10 (C, D), the content of malondialdehyde (MDA) and the degree of cell death in the transgenic overexpression lines were lower than those of the wild type, and the cell damage was smaller. These evidences show that the residual amount of active oxygen accumulated in the transgenic lines is less than that of the control lines after being subjected to drought stress, and then shows that the overexpressed PbrERF109 gene can effectively enhance the active oxygen scavenging ability of transgenic plants, thereby improving the plant's drought resistance.

In order to evaluate the drought resistance of PbrERF109 transgenic pear overexpression, under normal growth conditions, there was no significant difference in the morphology of the control and overexpression lines. After 20 days of drought treatment, it was found that the wild type was more severely wilted and water-stained than the transgenic line; Resuming watering and growing for 7 days, the two transgenic lines recovered quickly, but most of the control lines could not return to normal growth and turned yellow, and the terminal buds had died. The above results indicated that the drought resistance of the transgenic lines was stronger than that of the wild type.

It can be concluded from the above examples that the present invention transfers the drought-resistant transcription factor PbrERF109 into tobacco and Qiuzi pear, and the drought-resistant ability of the obtained transgene overexpression line has been greatly improved compared with the control wild type, and the transgene overexpression of tobacco The content of hydrogen peroxide and malondialdehyde in the strain is lower than that of the wild type, the residual reactive oxygen species in the plant is lower, and the cell damage is smaller, thereby improving the drought resistance of tobacco and Qiuzi pear plants.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Nanjing Agricultural University

<120> A Drought Resistant Transcription Factor PbrERF109, Its Preparation Method, Application, Encoded Protein and Application

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gtctacaatg ccacgtcatc cgtctcgacg agcggcaccc agttggcagc gggccaacca 180

gcacaacagg acaactactt ctcgccatcg ttgccgaatc aaaacaggaa ccagcaactg 240

agtttgggaa ccgggtttgt cgggatgaat gcgccaacta cgaggaagag caagaacaag 300

tacaggggcg tcaggcagag gccgtggggg aaatgggcgg cggagattcg agaccacga 360

cgggcggcga gggtgtggct agggacgttc gagacggcgg aggacgcggc cagggcttac 420

gacaaggccg ccgtcgagtt ccgcggaaat aaggcaaagc tcaatttccc atcggacccg 480

ggcggtcaca ttgtcacgac taacgacagt tctagtagtg gaactagtgc taatgccagt 540

attaatccag gattaattaa taagcaaaag caaaagaata ttagcgaaat tggggtcatg 600

gagaaggagg aggagaaggt tgatcaggtc aaactcaacc aggcgacgca gccggagaat 660

atgcaactgg gggtggtggc gaccgcggag agcagcgttg gtcatgagga ggatgaccag 720

ttcttgttgt gggacaatgg ctggctccga gatggtgaag atgacgactt aatggcatgg 780

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Met Val Ser Ala Leu Lys His Val Ile Ser Gly Gly Ser Ile Ser Gly

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Pro Thr Pro Gln Pro Met Pro Ala Val Tyr Asn Ala Thr Ser Ser Ser Val

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Asn Tyr Phe Ser Pro Ser Leu Pro Asn Gln Asn Arg Asn Gln Gln Leu

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Ser Leu Gly Thr Gly Phe Val Gly Met Asn Ala Pro Thr Thr Arg Lys

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Ser Lys Asn Lys Tyr Arg Gly Val Arg Gln Arg Pro Trp Gly Lys Trp

100 105 110

Ala Ala Glu Ile Arg Asp Pro Arg Arg Ala Ala Arg Val Trp Leu Gly

115 120 125

Thr Phe Glu Thr Ala Glu Asp Ala Ala Arg Ala Tyr Asp Lys Ala Ala

130 135 140

Val Glu Phe Arg Gly Asn Lys Ala Lys Leu Asn Phe Pro Ser Asp Pro

145 150 155 160

Gly Gly His Ile Val Thr Thr Asn Asp Ser Ser Ser Ser Gly Thr Ser

165 170 175

Ala Asn Ala Ser Ile Asn Pro Gly Leu Ile Asn Lys Gln Lys Gln Lys

180 185 190

Asn Ile Ser Glu Ile Gly Val Met Glu Lys Glu Glu Glu Lys Val Asp

195 200 205

Gln Val Lys Leu Asn Gln Ala Thr Gln Pro Glu Asn Met Gln Leu Gly

210 215 220

Val Val Ala Thr Ala Glu Ser Ser Val Gly His Glu Glu Asp Asp Gln

225 230 235 240

Phe Leu Leu Trp Asp Asn Gly Trp Leu Arg Asp Gly Glu Asp Asp Asp

245 250 255

Leu Met Ala Trp Leu Ser Thr Asn

260

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gggtnacccc tagttcgtgg ataacca 27

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agccggagaa tatgcaactg 20

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tctagaatgc ccttccatgc gaatcggata 30

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Claims (9)

1. A drought-resistant transcription factor PbrERF109, which has the nucleotide sequence shown in ESQ ID No.1. 2. The protein encoded by the drought-resistant transcription factor PbrERF109 according to claim 1 has the amino acid sequence shown in SEQ ID No.2. 3. the preparation method of the drought-resistant transcription factor PbrERF109 described in claim 1, comprises the following steps: Using pear cDNA as a template, the drought-resistant transcription factor PbrERF109 was obtained by PCR amplification with transcription factor primers; The transcription factor primer pair includes a transcription factor upstream primer and a transcription factor downstream primer, the transcription factor upstream primer has the nucleotide sequence shown in SEQ ID No.3, and the transcription factor downstream primer has the nucleotide sequence shown in SEQ ID No.4 The nucleotide sequence shown. 4. The preparation method according to claim 3, characterized in that, the system used in the PCR amplification comprises per 50 μl: 100ng pear cDNA, 10 μl Q5 Reaction Buffer, 1 μl 10mM dNTP, 0.5 μl Taq polymerase, 2.5 μl 10 μM Transcription factor upstream primer, 2.5 μl 10 μM transcription factor downstream primer, the balance is ddH ₂ O; The PCR amplification conditions include: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles; and extension at 72°C for 2 minutes after the cycle is completed. 5. The application of the drought-resistant transcription factor PbrERF109 according to claim 1 or the drought-resistant transcription factor PbrERF109 prepared by the preparation method according to any one of claims 3 to 5 or the protein according to claim 2 in improving plant drought resistance. 6. The use according to claim 5, wherein the plant comprises tobacco or pear. 7. application according to claim 6, is characterized in that, when described plant is tobacco, the method for described improving the drought resistance ability of tobacco comprises the following steps: connecting the drought-resistant transcription factor PbrERF109 with the pEASY-T1 cloning vector to obtain a recombinant vector; Transforming the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; The recombinant Agrobacterium was used to infect tobacco. 8. The application according to claim 7, characterized in that, every 5 μl of the total system connected with the pEASY-T1 cloning vector comprises: 4 μl of drought-resistant transcription factor PbrERF109 and 1 μl of pEASY-T1 cloning vector, the drought-resistant transcription factor PbrERF109 and the carrier The molar ratio is 3:1; The connection conditions include: reacting at 25° C. for 30 minutes. 9. The application according to claim 5, characterized in that, when the plant is a pear, the method for improving the drought resistance of the pear comprises: using a transient transformation method to transfer the drought-resistant transcription factor PbrERF109 into the pear .