阅读说明：本技术 一种小麦穗分枝基因dna序列扩增方法、分枝基因及应用 (Wheat ear branch gene DNA sequence amplification method, branch gene and application ) 是由 蔡华 赵荣 赵维萍 于 2020-07-24 设计创作，主要内容包括：本发明公开了一种小麦穗分枝基因DNA序列扩增方法、分枝基因及应用,以分枝小麦、未分枝小麦、中国春、小白芒四个品种的小麦叶片为试验材料,采用同源克隆的方法,在四种小麦中分别扩增出tb1基因全长。而对四种小麦tb1基因分析得知,该基因编码区全长1059bp,共编码352个氨基酸,通过验证确为分枝基因,该基因参与了小麦穗分枝的调控。本发明为培育出更多优良的小麦品种,丰富育种资源,进而培育出具有优良的农艺性状、高产抗倒伏的作物新品种提供基础,同时为作物分子遗传育种和性状改良、种质创新提供较好的科学基础。(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.)

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.

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 × EasySupermix, 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.