CN111808926A - A kind of wheat panicle branch gene DNA sequence amplification method, branch gene and application - Google Patents

Abstract

The invention discloses a method for amplifying a DNA sequence of a wheat ear branch gene, a branch gene and application thereof, wherein the full length of a tb1 gene is amplified in four kinds of wheat respectively by taking wheat leaves of four kinds of wheat, namely branched wheat, non-branched wheat, Chinese spring and awn as test materials and adopting a homologous cloning method. The analysis of four wheat tb1 genes shows that the total length of the coding region of the gene is 1059bp, 352 amino acids are coded, the gene is confirmed to be a branched gene by verification, and the gene participates in the regulation and control of wheat ear branching. The method provides a foundation for cultivating more excellent wheat varieties and enriching breeding resources, further cultivating new crop varieties with excellent agronomic characters and high yield and lodging resistance, and simultaneously provides a better scientific foundation for molecular genetic breeding, character improvement and germplasm innovation of crops.

Description

A kind of wheat panicle branch gene DNA sequence amplification method, branch gene and application

technical field

The invention relates to the technical field of genetic engineering, in particular to a method for amplifying a DNA sequence of a branching gene of wheat ears, a branching gene and application thereof.

Background technique

So far, wheat is a type of food crop that is widely planted in most parts of the world, providing most of the energy consumption basis for all human beings. Therefore, increasing the output of wheat is the basis for stabilizing the global economy, and it is also the sustainable and healthy national economy. important development goals. The yield of wheat is closely related to the fruiting part of the wheat. Studies have shown that among the three elements of wheat yield, the number of wheat ears plays a major role. Therefore, the research on the morphological development of wheat ears is not only beneficial to people's theoretical understanding of wheat ear development, but also to high-yield breeding of wheat.

Wheat ears play an important role in wheat development and branching traits. The spike-branched wheat increases the number of grains per spike through the formation of branch-cob on the main cob node, and increases the yield of wheat with larger spikes and more grains. The genes that control wheat panicle branching traits have not yet been clearly identified. Cloning and localization of related genes controlling branching in wheat can not only clarify the theoretical basis for controlling branching development in wheat, but also be applied to high-yield breeding of wheat.

In the process of development and formation of panicle branching in wheat, the formation and development of panicle branch may be involved in the formation and development of wheat panicle. To explore the regulatory mechanism of key genes regulating the formation of wheat side shoots, the control of panicle branching in branched wheat The location study and cloning analysis of branch genes are of great theoretical and practical significance, which are crucial to further understanding the branch panicle formation in branched wheat, and also open a road to the green revolution for many wheat breeders. door.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the above-mentioned prior art, the present invention provides a DNA sequence amplification method and application of a wheat ear branching gene, and conducts localization research and cloning analysis on the gene that controls ear branching in branched wheat, in order to cultivate more excellent It provides the basis for the development of new crop varieties with excellent agronomic traits, high yield and lodging resistance, and provides a good scientific basis for the genetic breeding, trait improvement and germplasm innovation of crop molecules.

In order to achieve the above-mentioned purpose, the technical scheme adopted in the present invention is: a method for amplifying the DNA sequence of a branch gene of wheat ear, comprising the following steps:

(1) Extracting genomic DNA and total RNA from wheat of a variety of different varieties, and designing gene-specific primers according to the bd1 gene and tb1 gene in Poaceae that have been disclosed by GenBank;

(2) According to the extracted genomic DNA and cDNA as templates, use bd1 gene and tb1 branch gene primers to carry out PCR amplification;

(3) clone and sequence the PCR amplification result, carry out the homology comparison of the sequenced results or design gene-specific primers according to the amplified branch gene as a template to carry out RT-PCR reaction, sequence after electrophoresis detection, and analyze step ( 2) Whether the gene obtained by PCR amplification is a wheat panicle branch gene.

Preferably, the various varieties of wheat in step (1) include at least branched wheat, unbranched wheat, Chinese spring, and small white awn.

Preferably in any of the above-mentioned schemes, the grasses in step (1) are maize, and according to the bd1 gene GenBank: AY178191.1 and tb1 gene GenBank: U94494.1 in maize, a Primer 5.0 software is used to design a UTR region at both ends. Gene-specific primers.

Preferably in any of the above solutions, the designed gene-specific primer sequences are shown in SEQ NO.1-SEQ NO.4 in the sequence listing.

Preferably in any of the above-mentioned schemes, the designed gene-specific primer sequence information is

SuperMix，补加ddH₂O至10uL体系。Preferably in any of the above-mentioned schemes, in step (2), the extracted genomic DNA and cDNA during PCR amplification are templates, bd1 gene and tb1 branch gene primers, prepare 10uL PCR system: 1uL template, each upstream and downstream primers. 0.5uL, 5uL Trans 2×Easy

SuperMix, supplemented with _ddH2O to 10uL system.

Preferably in any of the above-mentioned schemes, step (3) is also included after step (4), that is, after obtaining the full length of the branched gene, its maximum open reading frame is analyzed, thereby obtaining the corresponding amino acid sequence of the gene sequence. , and analyzed the amino acid sequence to obtain its conserved region in the evolutionary process. The phylogenetic tree was constructed using NCBI and DNAMAN software to analyze the distance between branch genes in wheat and other plants in the process of species evolution.

The present invention also provides a specific PCR primer composition designed based on the full-length cDNA sequence of the wheat panicle branching gene, and the primer sequences are the sequences shown in SEQ NO.5 to SEQ NO.6 in the sequence table. The primer sequence of SEQ NO.5 is TB1-F2: 5'-AGGACGGCTCCAGCAGCCTCT-3', and the primer sequence of SEQ NO.6 is TB1-R2: 5'-GTGCGGGAGTAGTTCTAATACCGT-3', which was synthesized by Shanghai Yingjun Biological Co., Ltd. .

The present invention also provides the full-length DNA sequences of the tb1 genes of the four types of wheat: branched wheat, unbranched wheat, Chinese spring and Xiaobaimang, the sequence numbers are KU847345, KU847346, KU847347, KU847348, and the gene sequences are shown in SEQNO in the sequence table .7, SEQ NO.8, SEQ NO.9, SEQ NO.10.

The present invention also provides the amino acid encoded by the above-mentioned wheat panicle branch gene DNA sequence.

The invention also provides the application of the above-mentioned DNA sequence of the wheat ear branching gene in screening the molecular breeding of wheat ear branching and analyzing the distance between the wheat branching gene and other plants in the species evolution process.

The present invention also provides the application of the above-mentioned wheat panicle branch gene in preparing branch gene wheat.

beneficial effect

Branching genes are key genes that regulate the branching mutation of wheat panicle traits. In order to study the function of wheat branching gene and its mechanism of action on wheat panicle branching, the present application takes the wheat leaves of four varieties of branched wheat, unbranched wheat, Chinese spring and Xiaobaimang as test materials, and adopts homologous wheat leaves. The cloning method was used to amplify the bd1 branch gene fragment and the full length of the tb1 gene in four kinds of wheat. The analysis shows that the bd1 gene does not match the expected results of the experiment, and the analysis of the four wheat tb1 genes shows that the coding region of the gene is 1059 bp in length and encodes a total of 352 amino acids. Branch regulation.

The invention provides a DNA sequence amplification method and application of a wheat panicle branching gene, and conducts localization research and cloning analysis on the gene that controls panicle branching in branched wheat. Cultivate new crop varieties with excellent agronomic traits, high yield and lodging resistance, and provide a good scientific basis for the genetic breeding of crop molecules, trait improvement, and germplasm innovation.

Description of drawings

Figure 1 is a comparison of the wheat spike shape of four different varieties, 1 is branched wheat; 2 is unbranched wheat; 3 is Chinese spring; 4 is Xiaobaimang;

Fig. 2 is the detection result of the extracted wheat genomic DNA and total RNA gel electrophoresis;

Figure 3 is the amplification results of specific primers, M is 2000bp Marker; 1 is branched wheat; 2 is unbranched wheat; 3 is Chinese spring wheat; 4 is Xiaobaimang;

Fig. 4 is a sequence alignment diagram of wheat ear branching gene (fragment);

Figure 5 shows the amplification results of tb1 gene-specific primers in cDNA and DNA, M is 2000bp Marker; 1 is branched wheat; 2 is unbranched wheat; 3 is Chinese spring wheat;

Fig. 6 is four kinds of wheat panicle branches tb1 gene sequence alignment diagram;

Figure 7 is a molecular evolutionary tree diagram of different plant TB1 homologous proteins;

Figure 8 is the full-length cDNA sequence and the encoded amino acid sequence of the branched wheat tb1 gene, wherein the marked ATG is the start codon, TGA is the stop codon, and the squares are the SP region, the TCP region and the R region of the amino acid sequence in turn .

Detailed ways

The present invention will be further described below.

Example 1

In order to achieve the above object, the technical scheme adopted in the present invention is:

The present invention provides a DNA sequence amplification method of wheat panicle branching gene: the detailed method is as follows: 1. Materials

(1.1) Test material

Common wheat varieties "China Spring" and "Xiaobaimang" were selected from the micro-core germplasm of Chinese wheat; "branched wheat" and "unbranched wheat" were large spike mutants of "Zhengmai 9023", "Zhengmai 9023" "Branch wheat" is a branched panicle mutant of "Zhengmai 9023", and the experimental materials were all planted in the Huashan wheat experimental base of Chuzhou University. At the beginning of April every year, the young leaves and young panicle tissues of four wheat varieties were collected when they were just heading. The comparison of the four wheat ear shapes is shown in Figure 1. The leaf DNA and total RNA of the panicle tissues of each variety were extracted and extracted. The genomic DNA and total RNA were stored at -20°C and -80°C, respectively.

(1.2) Test reagents

SuperMix，灭菌超纯水；琼脂糖凝胶电泳：琼脂糖、0.5×TBE缓冲液、Marker，核酸染液；胶回收试剂盒等。DNA and RNA extraction: RNAiso Plus, Reverse Transcription Reagent, C ₂ H ₅ OH, EasyPure Plant Genomic DNAKit; primers, Trans 2×Easy

SuperMix, sterile ultrapure water; agarose gel electrophoresis: agarose, 0.5×TBE buffer, Marker, nucleic acid staining solution; gel recovery kit, etc.

(1.3) Test equipment

Refrigerated high-speed centrifuge (Anhui Jiawen), ultra-low temperature refrigerator (Thermo Fishery), clean operating table (Suzhou), micropipettes and large, medium and small tips, PCR reactor, gel electrophoresis UV analyzer, metal bath, palm low speed Centrifuges, analytical balances, autoclaves, etc.

2 Test methods

(2.1) Extraction of genomic DNA and total RNA

Refer to the instruction manual of the EasyPure Plant Genomic DNA Kit produced by Transgene, take four different varieties of wheat young leaves in a mortar that has been sterilized by high temperature in advance, add liquid nitrogen and quickly grind to powder, and transfer the powder to a sterilizer. After the bacteria were frozen in a 1.5mL centrifuge tube, the genomic DNA was extracted according to the operation steps of the kit, and the extracted DNA samples were stored in a -20°C refrigerator for future use.

The extraction of total genomic RNA was carried out according to the extraction steps of RNA extraction lysate produced by TaKaRa Company. The extracted total genomic RNA of four kinds of wheat panicle tissues was stored in a -80℃ refrigerator for future use. The extracted leaf genomic DNA and panicle tissue total RNA of branched wheat, unbranched wheat, Chinese spring, and small white awn wheat were detected by gel electrophoresis, and the results are shown in Figure 2.

(2.2) Reverse transcription of total genomic RNA

According to the operation steps of the reverse transcription kit produced by Tiangen Biotechnology Co., Ltd., the operation was performed: 5ug total genomic RNA, 2μL oligo(dT) Primer (50pmol/L) primers were sequentially added to the sterilized PCR tube, and RNase- Make up freeWater to 14.5 μL; heat at 70°C for 5 min, immediately after the reaction, cool on ice and centrifuge for 30 s. Add 4uL 5×First-Strand Buffer (containing DTT), 0.5uL RNasin, and 1uL (200U) TRANScript M-MLV in sequence on ice, mix well with a pipette, and react at 25°C for 10min. React at 42°C for 50 min; react at 95°C for 5 min to terminate the reaction. After the product was diluted 10 times, the quality of reverse transcription was detected with the internal reference gene, and the subsequent PCR amplification test was carried out.

(2.3) Design of branched gene-specific primers

According to relevant literature, both bd1 and tb1 genes in maize belong to a class of key genes regulating maize branching, and also play an important role in the formation of maize branching. At present, the cloning of this gene is mainly concentrated in Poaceae, and there is no research on the cloning of wheat bd1 gene and tb1 homologous gene. Therefore, according to the conservation of the bd1 gene (GenBank: AY178191.1) and the tb1 gene (GenBank: U94494.1) in maize, a pair of gene-specific primers were designed in the UTR regions at both ends using Primer 5.0 software, and amplified by long fragment PCR its entire coding region. The sequence information of the designed primers is shown in Table 1 below, and all primers were synthesized by Shanghai Yingjun Biotechnology Company.

Table 1 Primer names and sequences

Table 1 the name and sequence of the primer

(2.4) PCR amplification of target gene

SuperMix，补加ddH₂O至10uL体系。以bd1基因为引物的一组PCR反应程序为94℃预变性3min；94℃变性30s，56℃退火30s，72℃延伸1min共35个循环；72℃10min；以tb1基因为引物的一组PCR反应程序：94℃3min；94℃30s，55℃30s，72℃1min共35循环；72℃10min结束反应。(2.4.1)小麦bd1基因特异性引物PCR扩增According to the extracted genomic DNA and cDNA as templates, primers for bd1 gene and tb1 branch gene, prepare a 10uL PCR system: 1uL template, 0.5uL upstream and downstream primers, 5uL Trans 2×Easy

SuperMix, supplemented with _ddH2O to 10uL system. A set of PCR reaction procedures with bd1 gene as primers were as follows: 94°C pre-denaturation for 3 min; 35 cycles of denaturation at 94°C for 30s, 56°C annealing for 30s, and 72°C extension for 1 min; 72°C for 10 min; a set of PCR with tb1 gene as primers Reaction program: 3 min at 94°C; 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 1 min; complete the reaction at 72°C for 10 min. (2.4.1) PCR amplification of wheat bd1 gene specific primers

BD-R1 and BD-F1 were used as specific primers to carry out PCR amplification with template DNA and cDNA respectively. The results showed that no results were detected in the system using cDNA as the template, indicating that the bd1 gene was not involved in the panicle tissue of wheat at heading stage. Express. The amplification results in the DNA are shown in Figure 3; the amplified target band was purified and sent to Shanghai Sangon Biological Co., Ltd. for sequencing, and a 248bp sequence was obtained.

Alignment and Analysis of Amplified Sequences of bd1 Gene

The blast comparison in NCBI showed that the four sequences had the highest homology of 94% with a sequence on chromosome 3B of wheat (GenBank: HG670306.1). There are differences in the amplified sequences of unbranched wheat species. As shown in Figure 4, the aligned sequences are branched wheat, unbranched wheat and maize bd1 sequences, and the homology of the alignment results is 56.55%. From the comparison result of 248bp of bd1 gene of panicle branch, it does not meet the expected result of the experiment.

(2.4.2) PCR amplification of wheat tb1 gene specific primers

Using TB1-R1 and TB1-F1 as a pair of gene-specific primers, PCR amplification was carried out with cDNA and DNA as templates respectively. The results of PCR products detected by gel electrophoresis are shown in Figure 5 below; according to the amplified electrophoresis results, it can be seen that When DNA and cDNA are used as templates, the amplified length is about 1000 bp. The amplified target band is cloned by TA, and the samples are sent to Shanghai Huajin Biological Co., Ltd. for sequencing. The cDNA sequences of 1199bp, 1165bp, 1186bp and 1191bp and the DNA sequences of 1119bp, 1122bp, 1124bp and 1115bp were obtained from branched wheat, unbranched wheat, Chinese spring and Xiaobaimang respectively.

Sequence alignment analysis of wheat tb1 gene

After PCR reaction-sampling sequencing-result comparison analysis, the DNA sequencing lengths of tb1 gene in four kinds of wheat were 1119bp, 1122bp, 1124bp, 1115bp respectively, and the similarity was 88.64% after DNAMAN sequence comparison. The comparison results are shown in Figure 6 below: wherein, WF is unbranched wheat, FZ is branched wheat, CS is Chinese spring, BM is Xiaobaimang, and * is the same sequence. After blast comparison by NCBI, it was found that the similarity with the tb1 gene of ryegrass from the family Poaceae was 83%, the similarity with the tb1 gene in rice was as high as 80%, and the similarity with the tb1 gene in maize was 70%. The gene is the tb1 gene in wheat. The four sequences were submitted to NCBI for registration, and the serial numbers of the tb1 gene sequences of the four types of wheat: branched wheat, unbranched wheat, Chinese spring and Xiaobaimang were KU847345, KU847346, KU847347, KU847348, and branched wheat and unbranched wheat were obtained. There are individual base differences between branched wheat, so it can be speculated that the tb1 gene may be the key gene that causes wheat panicle branching. Due to the mutation of individual bases in the branching gene, the original branching trait of wheat is mutated to non-branching. branching traits.

(2.5) Phylogenetic relationship between tb1 gene and TB1 homologous genes of other grasses

In order to further clarify the function of tb1 gene and understand its phylogenetic relationship with TB1 homologous genes of other grasses, we selected rice (GenBank: AY286002.1), African millet ( GenBank: AY631857.1), early bamboo (GenBank: DQ842223.1), ryegrass (GenBank: GU987123.1), maize (GenBank: U94494.1) and other grasses The TB1 protein sequences of wheat species were compared and analyzed, and the comparison results showed that the amino acid sequences in their conserved regions were basically the same, with an overall similarity of 67.7%, and they were divided into three conserved evolution regions, namely SP region, TCP region, The R regions belong to the Poaceae TCP family of transcription factors.

Molecular phylogenetic tree analysis of different plant TB1 homologous proteins is shown in Figure 7. The full-length cDNA sequence and the encoded amino acid sequence of the branched wheat tb1 gene are shown in Figure 8. The amino acid sequences of the four wheat tb1 genes were compared in NCBI with blastp. The results showed that the amino acids encoded by the four wheat tb1 genes The sequence similarity with the TCP domain protein in wheat was up to 87%, and the sequence similarity with the TCP transcription factor protein and TB1 protein was 87% and 86%, respectively. According to relevant literature, the tb1 gene in the grasses maize and rice share some common regulatory elements, such as the TCP transcription factor. It can be seen that the amplified sequence in the four types of wheat is indeed the tb1 gene, which belongs to the same protein family as maize and rice and other gramineous plants, and plays a regulatory role in the process of branching traits in wheat ears. (2.5.1) PCR Amplification Results Cloning and Sequencing

The products after PCR amplification were subjected to gel cutting purification and the purified samples were sent to Sangon Biology (Shanghai) Co., Ltd. for sequencing.

(2.5.2) Bioinformatics analysis of sequencing results

The sequencing results were compared for homology using the Alignment multiple sequence alignment of DNAMAN software, and BLAST analysis was performed on NCBI to analyze whether the gene obtained by PCR amplification was a wheat panicle branch gene. After obtaining the full length of the branched gene, its largest open reading frame (Open Reading Frame, ORF) is analyzed, thereby deriving the amino acid sequence corresponding to the gene sequence, and analyzing the amino acid sequence to obtain its evolutionary process. in the conserved region. The phylogenetic tree was constructed using NCBI and DNAMAN software to analyze the distance between branch genes in wheat and other plants in the process of species evolution.

According to the amplified tb1 gene in branched wheat as template, a pair of gene-specific primers were designed for RT-PCR reaction. After electrophoresis detection, the samples were purified and sent for sequencing. The sequencing results showed that 573bp cDNA fragments were amplified in different varieties of wheat. , the amplified cDNA fragment is indeed the tb1 gene by sequencing-sequence alignment analysis. It is of great significance to study the key genes that regulate the branching of wheat panicle traits for the future use of molecular breeding methods to accurately improve wheat yield and fine varieties. The present application clones the key gene that regulates the branching of the wheat panicle, preliminarily analyzes the expression and regulation of the branching gene, understands the action mechanism of the branching gene, and provides basic information for breeders.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Substitutions should be covered within the protection scope of the present invention.

sequence listing

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cgtcgacggc aagcacaagc cgccagggac ggaggctgga ggtggtgatc acggcgaggg 600

gaagaagcca gtgccaaggg cagccagaag ggcacccgcc aatccaaaac cgcaaaggaa 660

attggccagt gcgcacccga tccccgacaa ggagtcgagg acgaaggccc gggagagggc 720

gagggagcgg acgagggaaa agaaccggat gcgatgggtg acgctcgcgt ccacaattaa 780

catcgagccg gcagccaccg gcatggcggc ggcgagggcg aggcgagttg gtcacgaatc 840

cgaacaattt gatctatcgc tcctcgtcca tgaacacatc aagagctgaa ttggaggagg 900

ggtgctcgtc gtccatgccg agcgaagcga tcatgactgg cttcggctat ggagggtacg 960

gaagcagcgg caactactac cagtatcagc tggagcagca atgggagctc ggtggcagtg 1020

gtgtttacca actcgcagtc cctactgata atgccgcgac atgccgtacg gtatga 1076

Claims (9)

1. A wheat ear branch gene DNA sequence amplification method is characterized by comprising the following steps:

(1) extracting genome DNA and total RNA from wheat of different varieties, and designing gene specific primers according to bd1 gene and tb1 gene in gramineous plants published by GenBank;

(2) performing PCR amplification by using bd1 gene and tb1 branched gene primers according to the extracted genomic DNA and cDNA as templates;

(3) and (3) cloning and sequencing the PCR amplification result, carrying out homology comparison on the sequencing result or designing a gene specific primer according to the amplified branch gene as a template to carry out RT-PCR reaction, carrying out sequencing after electrophoresis detection, and analyzing whether the gene obtained by PCR amplification in the step (2) is the wheat ear branch gene or not.

2. The method of claim 1, wherein the wheat of the plurality of different varieties in step (1) comprises at least branched wheat, unbranched wheat, Chinese spring, and Miscanthus sinensis.

3. The method for amplifying the DNA sequence of a branched wheat ear gene of claim 1, wherein the gramineae in the step (1) is maize, and the DNA sequence of the branched wheat ear gene is obtained by using the gene GenBank of bd1 gene AY178191.1 and tb1 gene GenBank: u94494.1, a pair of gene-specific primers was designed in the UTR region at both ends using Primer 5.0 software.

4. The method for amplifying the wheat ear branch gene DNA sequence of claim 1, wherein the gene specific primer sequence in step (1) is shown in SEQ NO.1-SEQ NO.4 of the sequence list.

5. A specific PCR primer composition designed based on the whole length of a wheat ear branch gene cDNA sequence is characterized in that primer sequences are respectively sequences shown as SEQ NO.5 to SEQ NO.6 in a sequence table.

6. A wheat ear branch gene DNA sequence is disclosed, the wheat is branch wheat, non-branch wheat, Chinese spring and awn, the branch gene is tb1 gene, the tb1 gene sequence of branch wheat, non-branch wheat, Chinese spring and awn is shown in SEQ NO.7, SEQ NO.8, SEQ NO.9 and SEQ NO.10 in the sequence list.

7. Amino acid coded by wheat ear branch gene DNA sequence.

8. Use of the branched wheat ear gene DNA sequence obtained by the method according to any one of claims 1 to 4 for screening branched wheat ear molecular breeding, analyzing the distance of the genetic relationship between the branched wheat ear gene and other plants in the species evolution process.

9. Use of the branched wheat head gene obtained by the method of any one of claims 1 to 4 for the preparation of branched gene wheat.

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