CN111808926A

CN111808926A - Wheat ear branch gene DNA sequence amplification method, branch gene and application

Info

Publication number: CN111808926A
Application number: CN202010728388.2A
Authority: CN
Inventors: 蔡华; 赵荣; 赵维萍
Original assignee: Chuzhou University
Current assignee: Chuzhou University
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2020-10-23

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

Wheat ear 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 wheat ear branching gene DNA sequence amplification method, a branching gene and application.

Background

So far, wheat is a food crop which is widely planted in most of the world, provides most of energy consumption foundation for the whole human, improves the yield of wheat, is the foundation for stabilizing the global economy, and is also an important target for the sustainable and healthy development of national economy. The yield of wheat is closely related to the fructification site of wheat. The research shows that the number of wheat ears plays a main role in the three elements of the wheat yield, so that the research on the morphological development of the wheat ears is beneficial to the theoretical understanding of people on the development of the wheat ears and the high-yield breeding of wheat.

Wheat ears play an important role in wheat development and formation of branched traits. The branched wheat has the advantages that the branched cobs are formed on the main cob joints to increase the grain number of the wheat per ear, and the grains per ear are large, so that the yield of the wheat is improved. No specific location exists for the gene related to controlling the branching traits of wheat ears. The related gene for controlling wheat branching is cloned and positioned, so that not only can the theoretical basis for controlling wheat branching development be clarified, but also the gene can be applied to high-yield breeding of wheat.

In the development and formation process of ear branches of wheat with branch ears, the formation and development of the side branches at the ear parts of the wheat can be related, the regulation mechanism of key genes for regulating the formation of the side branches of the wheat is discussed, the positioning research and the clone analysis of the genes for controlling the ear branches of the branched wheat have very important theoretical significance and practical significance, the genes are important for further understanding the formation of the branch ears of the wheat with branch ears, and meanwhile, the door leading to the green revolution is opened for numerous wheat breeding researchers.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a wheat ear branch gene DNA sequence amplification method and application, which are used for carrying out positioning research and cloning analysis on the gene for controlling ear branches of the branched wheat, so that a foundation is provided for cultivating more excellent wheat varieties, enriching breeding resources, further cultivating new crop varieties with excellent agronomic characters and high yield and lodging resistance, and a better scientific foundation is provided for genetic breeding, character improvement and germplasm innovation of crop molecules.

In order to achieve the purpose, the invention adopts the technical scheme that: a wheat ear branch gene DNA sequence amplification method comprises 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.

Preferably, the wheat of the plurality of different varieties in step (1) at least comprises branched wheat, unbranched wheat, Chinese spring and awn.

In any of the above schemes, preferably, the gramineae in the step (1) is maize, and the genes according to the bd1 gene GenBank: 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.

In any of the schemes, preferably, the designed gene-specific primer sequence is shown in SEQ NO.1-SEQ NO.4 of the sequence table.

In any of the above embodiments, the sequence information of the designed gene-specific primers is preferably

In any of the above embodiments, in step (2), the genomic DNA and cDNA extracted during PCR amplification are used as templates, and bd1 gene and tb1 branched gene primers are used to prepare a 10uL PCR system: 1uL template, upstream and downstream primers 0.5uL, 5uLTrans 2 × Easy

Supermix, supplemented with ddH₂O to 10uL system.

In any of the above schemes, step (3) preferably further comprises step (4) of analyzing the maximum open reading frame after obtaining the full length of the branched gene, thereby obtaining the amino acid sequence corresponding to the gene sequence, and analyzing the amino acid sequence to obtain the conserved region during the evolution process. The phylogenetic tree is constructed by using NCBI and DNAMAN software, and the distance of the genetic relationship between the branching gene in wheat and other plants in the species evolution process is analyzed.

The invention also provides a specific PCR primer composition designed based on the full length of the wheat ear branch gene cDNA sequence, and the primer sequence is shown as SEQ NO.5 to SEQ NO.6 in the sequence table. The primer sequence in SEQ NO.5 is TB 1-F2: 5'-AGGACGGCTCCAGCAGCCTCT-3', SEQ NO.6 the primer sequence is TB 1-R2: 5'-GTGCGGGAGTAGTTCTAATACCGT-3', handed in to Shanghai Junjun biology, Inc.

The invention also provides the full length of the tb1 gene DNA sequence of four kinds of wheat, including branched wheat, non-branched wheat, Chinese spring and aweto, with the sequence numbers of KU847345, KU847346, KU847347 and KU847348, and the gene sequences are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 in the sequence list.

The invention also provides the amino acid coded by the wheat ear branch gene DNA sequence.

The invention also provides the application of the wheat ear branch gene DNA sequence in screening wheat ear branch molecular breeding and analyzing the distance of the genetic relationship between the wheat ear branch gene and other plants in the species evolution process.

The invention also provides application of the wheat ear branch gene in preparing branch gene wheat.

Advantageous effects

The branching gene is a key gene for regulating the branching mutation of the spike trait of wheat. In order to research the function of the wheat branched gene and the action mechanism of the wheat branched gene on wheat ear branches, the method takes the wheat leaves of four varieties of branched wheat, non-branched wheat, Chinese spring and Miscanthus floridulus as test materials, and adopts a homologous cloning method to respectively amplify a bd1 branched gene segment and a tb1 full length gene in the four varieties of wheat. Analysis shows that the bd1 gene does not accord with the expected result of the test, while analysis of four wheat tb1 genes shows that the full length of the coding region of the gene is 1059bp, the coding region totally encodes 352 amino acids, and the gene is confirmed to be a branched gene through verification and participates in the regulation and control of wheat ear branches.

The invention provides a method for amplifying a wheat ear branch gene DNA sequence and application thereof, which carries out positioning research and clone analysis on the gene for controlling ear branches of branch wheat, provides a foundation for cultivating more excellent wheat varieties, enriching breeding resources, further cultivating new crop varieties with excellent agronomic characters and high yield and lodging resistance, and provides a better scientific foundation for genetic breeding, character improvement and germplasm innovation of crop molecules.

Drawings

FIG. 1 shows a comparison of the spike shapes of four different varieties of wheat, 1 being branched wheat; 2 is unbranched wheat; 3 is Chinese spring; 4 is Miscanthus floridulus;

FIG. 2 shows gel electrophoresis detection results of extracted wheat genome DNA and total RNA;

FIG. 3 shows the result of amplification of specific primers, M is 2000bp Marker; 1 is branched wheat; 2 is unbranched wheat; 3 is Chinese spring wheat; 4 is Miscanthus floridulus;

FIG. 4 is a diagram showing the sequence alignment of wheat ear branch genes (fragments);

FIG. 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; 4 is Miscanthus floridulus;

FIG. 6 is a diagram showing the alignment of the tb1 gene sequences of four wheat ear branches;

FIG. 7 is a molecular dendrogram of homologous proteins from different plant TB 1;

FIG. 8 shows the full-length cDNA sequence and the encoded amino acid sequence of the tb1 gene of branched wheat, in which the labeled ATG is the initiation codon, TGA is the termination codon, and the SP region, TCP region and R region of the amino acid sequence are in sequence in the box.

Detailed Description

The present invention is further described below.

Example 1

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a method for amplifying a wheat ear branch gene DNA sequence, which comprises the following steps: 1 Material

(1.1) test Material

Common wheat varieties of 'Chinese spring' and 'Xiaobaimisch' are all selected from Chinese wheat micro-core germplasm; the branched wheat and the non-branched wheat are spike mutants of Zheng wheat 9023 and the branched wheat is a spike mutant of Zheng wheat 9023, and the test materials are planted in the test base of Huashan wheat of Chuzhou academy. Respectively adopting young leaf and young ear tissues of four wheat varieties just after ear emergence every year at the beginning of four months, comparing the shapes of the four wheat ears as shown in figure 1, extracting DNA (deoxyribonucleic acid) of each variety of leaf and total RNA (ribonucleic acid) of each variety of ear tissues, and respectively storing the extracted genomic DNA and total RNA in an environment at-20 ℃ and-80 ℃.

(1.2) test reagents

DNA and RNA extraction: RNAioso Plus, reverse transcription reagent, C₂H₅OH, EasyPure Plant Genomic DNAkit kit; primer, Trans 2 × Easy

SuperMix, sterilizing ultrapure water; agarose gel electrophoresis: agarose, 0.5 XTBE buffer solution, Marker and nucleic acid staining solution; a gel recovery kit and the like.

(1.3) test apparatus

Freezing high-speed centrifuge (Anhui Jiawen), ultra-low temperature refrigerator (Sammerfo), clean operating platform (Suzhou), micro-pipetting gun, large, medium and small gun head, PCR reaction instrument, gel electrophoresis ultraviolet analyzer, metal bath, palm low-speed centrifuge, analytical balance, high-pressure steam sterilization pot, etc.

2 test method

(2.1) extraction of genomic DNA and Total RNA

Referring to the operation of an easy pure Plant Genomic DNA Kit produced by Transgene company, young leaves of four different varieties of wheat are taken and put into a mortar subjected to high-temperature sterilization in advance, liquid nitrogen is added into the mortar, the mixture is quickly ground into powder, the powder is transferred into a sterilized and frozen 1.5mL centrifuge tube, Genomic DNA extraction is carried out according to the operation steps of the Kit, and an extracted DNA sample is stored in a refrigerator at the temperature of-20 ℃ for later use.

The extraction of the total RNA of the genome is carried out according to the extraction procedure of RNA extraction lysate produced by TaKaRa company, and the extracted total RNA of the four wheatear tissue genomes is stored in a refrigerator at the temperature of minus 80 ℃ for later use. The extracted leaf genome DNA and ear tissue total RNA of branched wheat, unbranched wheat, Chinese spring and small white mango wheat are subjected to gel electrophoresis detection, and the results are shown in FIG. 2.

(2.2) reverse transcription of Total genomic RNA

The operation was carried out according to the procedure of reverse transcription kit manufactured by Tiangen biology, Ltd: 5ug of total genomic RNA, 2. mu.L of oligo (dT) Primer (50pmol/L) Primer, were sequentially added to the sterilized PCR tube and supplemented to 14.5. mu.L with RNase-freeWater; heating at 70 deg.C for 5min, immediately cooling on ice after reaction, and centrifuging for 30 s. 4uL of 5 XFirst-Strand Buffer (containing DTT), 0.5uL of RNase, 1uL (200U) of TRANScript M-MLV were added in this order on ice, mixed with a pipette and reacted at 25 ℃ for 10 min. Reacting at 42 ℃ for 50 min; the reaction was terminated by reacting at 95 ℃ for 5 min. After the product is diluted by 10 times, the reverse transcription quality is detected by using an internal reference gene, and a subsequent PCR amplification test is carried out.

(2.3) design of primers specific to branched genes

The reference of related documents shows that the bd1 gene and the tb1 gene in maize belong to a key gene for regulating maize branches, and play an important role in regulating the formation of the maize branches. At present, the cloning of the gene is mainly concentrated in gramineae, and no research is carried out on the cloning of the wheat bd1 gene and a tb1 homologous gene. Therefore, according to the conservation of the bd1 gene (GenBank: AY178191.1) and the tb1 gene (GenBank: U94494.1) in corn, a pair of gene-specific primers are designed in UTR regions at two ends by using Primer 5.0 software, and the whole coding region is amplified by long-fragment PCR. The sequence information of the designed primers is shown in Table 1 below, and all primers were synthesized by Shanghai Invitrogen Biotech.

TABLE 1 primer names and sequences

Table 1 the name and sequence of the primer

(2.4) PCR amplification of the target Gene

According to the extracted genomic DNA and cDNA as templates, bd1 gene and tb1 branched gene primers, a 10uLPCR system is prepared: 1uL template, upstream and downstream primers 0.5uL, 5uL Trans 2 × Easy

Supermix, supplemented with ddH₂O to 10uL system. One group of PCR reaction programs with the bd1 gene as a primer is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; 10min at 72 ℃; a set of PCR reaction programs with tb1 gene as primer: 3min at 94 ℃; 35 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; the reaction was terminated at 72 ℃ for 10 min. (2.4.1) PCR amplification of wheat bd1 Gene specific primers

The results of PCR amplification using the template DNA and cDNA as templates, respectively, with BD-R1 and BD-F1 as specific primers, were not detected in a system using cDNA as a template, indicating that the BD1 gene was not expressed in ear tissues at the heading stage of wheat. The results of the amplification in DNA are shown in FIG. 3; and purifying the amplified target band, and then sending the purified target band to Shanghai biological Limited company for sequencing to obtain a 248bp sequence.

Alignment analysis of bd1 Gene amplification sequences

The blast comparison result in NCBI shows that the homology of the four sequences and a sequence (GenBank: HG670306.1) on the chromosome 3B of wheat chromosome is 94% at most, DNAMAN comparison shows that the sequence amplified in branched wheat and the sequence amplified in non-branched wheat have difference, as shown in figure 4, the comparison sequences are branched wheat, non-branched wheat and maize bd1 sequences in sequence, and the homology of the comparison result is 56.55%. From the 248bp comparison result of the ear branch bd1 gene, the expected result of the test is not met.

(2.4.2) PCR amplification of wheat tb1 Gene specific primers

TB1-R1 and TB1-F1 are used as a pair of gene specific primers, cDNA and DNA are used as templates for PCR amplification respectively, and the detection result of the PCR product through gel electrophoresis is shown in the following figure 5; according to the amplified electrophoresis results, the amplified length is about 1000bp when the DNA and the cDNA are taken as templates, and the target band obtained by amplification is subjected to TA cloning and then sent to Shanghai Huajin biology Limited for sequencing. Obtaining the cDNA sequences of 1199bp, 1165bp, 1186bp and 1191bp and the DNA sequences of 1119bp, 1122bp, 1124bp and 1115bp in the branched wheat, the non-branched wheat, the Chinese spring and the awn respectively.

Alignment analysis of wheat tb1 gene sequence

Through PCR reaction, sample feeding sequencing and result comparison analysis, the DNA sequencing lengths of tb1 genes in the four kinds of wheat are 1119bp, 1122bp, 1124bp and 1115bp respectively, and after DNAMAN sequence comparison, the similarity is found to be 88.64%. The alignment results are shown in FIG. 6 below: wherein WF is unbranched wheat, FZ is branched wheat, CS is Chinese spring, BM is Miscanthus floridulus, and the sequences are the same. When the blast comparison is carried out by NCBI, the similarity of the gene with the tb1 gene of ryegrass of Gramineae is found to be 83 percent, the similarity of the gene with the tb1 gene of rice is up to 80 percent, and the similarity of the gene with the tb1 gene of corn is 70 percent, so that the amplified gene can be basically determined to be the tb1 gene in wheat. The four sequences are submitted to NCBI registration to obtain the sequence numbers of the tb1 gene sequences of the four wheat of branched wheat, non-branched wheat, Chinese spring and aweto which are KU847345, KU847346, KU847347 and KU847348 respectively, and the branched wheat and the non-branched wheat have individual base difference, so that the tb1 gene can be presumed to be a key gene causing wheat ear branching, and the wheat originally showing the branched character is mutated into the non-branched character due to the mutation of the individual base of the branched gene.

(2.5) phylogenetic relationship between TB1 gene and other gramineae TB1 homologous gene

In order to further clarify the function of TB1 gene and understand its phylogenetic relationship with other homologous genes of gramineous plant TB1, we selected several gramineous plant TB1 protein sequences of rice (GenBank: AY286002.1), African millet (GenBank: AY631857.1), early bamboo (GenBank: DQ842223.1), ryegrass (GenBank: GU987123.1), corn (GenBank: U94494.1) and so on according to the comparison of branched genes in NCBI, and compared the results with the amplified four kinds of wheat TB1 protein sequences, the comparison shows that the amino acid sequences in their conserved regions are basically consistent, the overall similarity is 67.7%, and the sequences are divided into three conserved evolutionary regions, namely SP region, TCP region and R region, all belonging to gramineous TCP family transcription factors.

Molecular phylogenetic tree analysis of homologous proteins of different plant TB1, as shown in FIG. 7. The full-length cDNA sequence and the coding amino acid sequence of the TB1 gene of the branched wheat are shown in figure 8, the amino acid sequences of the TB1 genes of four kinds of wheat are subjected to amino acid sequence comparison in blastp in NCBI, and the result shows that the similarity of the amino acid sequences coded by the TB1 genes of the four kinds of wheat and the sequence of a TCP structural domain protein in the wheat is up to 87 percent, and the similarity of the amino acid sequences coded by the TB1 genes of the four kinds of wheat and the sequence of a TCP structural domain protein in the wheat is 87 percent and 86 percent respectively. According to the literature, tb1 gene has some common regulatory elements, such as TCP transcription factor, in corn and rice, which are gramineae. Therefore, the sequence amplified from the four wheat is determined to be tb1 gene, and the tb1 gene belongs to a protein family with gramineae plants such as corn and rice and plays a role in regulating the process of branching character of wheat head. (2.5.1) sequencing by cloning the PCR amplification results

The products after PCR amplification were gel-cut and purified and the purified samples were sent to Bionics (Shanghai) Co., Ltd for sequencing.

(2.5.2) bioinformatic analysis of sequencing results

And carrying out homology comparison on the sequencing result by utilizing Alignment multiple sequence comparison of DNAMAN software, carrying out BLAST analysis on NCBI, and analyzing whether the gene obtained by PCR amplification is the wheat ear branch gene. After the full length of the branched gene is obtained, the maximum Open Reading Frame (ORF) of the branched gene is analyzed, so that an amino acid sequence corresponding to the gene sequence is derived, and the amino acid sequence is analyzed to obtain a conserved region of the branched gene in the evolution process. The phylogenetic tree is constructed by using NCBI and DNAMAN software, and the distance of the genetic relationship between the branching gene in wheat and other plants in the species evolution process is analyzed.

Designing a pair of gene specific primers to perform RT-PCR reaction according to tb1 gene in the amplified branched wheat as a template, purifying and sampling for sequencing after electrophoresis detection, wherein sequencing results show that 573bp cDNA fragments are amplified in different varieties of wheat, and sequencing-sequence comparison analysis shows that the amplified cDNA fragments are tb1 gene. The research and control of the key gene for the branching of the wheat ear character has important significance for accurately improving the yield and the excellent variety of wheat by utilizing a molecular breeding means in the future. The application clones a key gene for regulating the branching of the wheat spike, preliminarily analyzes the expression regulation condition of the branching gene, knows the action mechanism of the branching gene and provides basic information for breeding workers.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

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cgacaaggcc agcaagacgg tgcaatggct cctcgatacg tcaaagggcg ccatcaggga 480

ggtcatgact gacgaggcgt cctccgactg cgaggaggac ggctccagca gcctctccgt 540

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

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

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.