Application of BN1 gene in construction of multi-branch high-yield soybean

Abstract

The invention belongs to the technical field of soybean genetic breeding. More specifically, the invention relates to application of the BN1 gene in construction of multi-branch high-yield soybeans. The invention discloses an application of a BN1 gene in improvement of soybean yield. The BN1 gene is a BN1a gene and / or a BN1b gene. The sequence of the BN1a gene is as shown in SEQ ID NO. 1; and the sequence of the BN1b gene is as shown in SEQ ID NO. 3. The soybean bn1a-bn1b double mutant is obtained through a CRISPR / Cas9 method, and researches find that the number of branches of the soybean bn1a-bn1b double mutant is increased, the number of pods of a single plant is increased, the number of grains of the single plant is increased, and the grain weight of the single plant is increased. The BN1a gene and the BN1b gene are used for improving soybeans, and multi-branch high-yield soybean germplasm resources are created, so that the soybean yield is increased, and the BN1a gene and the BN1b gene have good application prospects and value in soybean breeding work.

Description

Technical Field

[0001] The present invention belongs to the technical field of soybean genetic breeding, and more specifically, relates to the application of BN1 gene in constructing multi-branched high-yield soybean. Background Art

[0002] As an important food and cash crop, soybean (Glycine max) is the main source of high-quality plant protein and edible oil. As the country of origin of soybean, my country is rich in soybean germplasm resources and has a long history of planting. However, due to many problems such as small planting area and low yield, the current total soybean production in my country is difficult to meet the people's growing consumption needs. According to relevant data from the National Bureau of Statistics, in 2020, my country's soybean production accounted for 15% of total consumption, and more than 80% of soybeans had to be imported from abroad. Therefore, improving soybean yield potential is an important task in soybean breeding.

[0003] The four elements that make up soybean yield include the number of plants per unit area, the number of pods per plant, the number of grains per plant, and the grain weight. In addition, the formation of branches can improve the utilization rate of light energy during soybean cultivation, thereby affecting the number of pods and grains per plant, and thus affecting the yield; and the development of soybean branches can play a certain compensatory role for the uneven emergence of seedlings during the planting process. The increase in branch yield can effectively make up for the yield loss caused by the low emergence rate. Therefore, finding the key genes for soybean branch development is of great significance to improving soybean yield. Summary of the invention

[0004] The present invention aims to overcome the shortage of soybean-related candidate gene resources in existing soybean branch number molecular breeding and provide a new application of the BN1 gene in constructing multi-branched high-yield soybeans.

[0005] The first object of the present invention is to provide application of BN1 gene.

[0006] The second object of the present invention is to provide the application of the BN1 gene fragment shown in SEQ ID NO.5.

[0007] The third object of the present invention is to provide a method for breeding high-yield soybean varieties.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention uses CRISPR / Cas9 method to target and knock out soybean BN1 gene (BN1a gene, BN1b gene) and successfully obtains soybean bn1a-bn1b double mutants, and studies the functions of soybean BN1a and BN1b genes in improving soybean yield. It is found that knocking out soybean BN1a and BN1b genes significantly increases the number of soybean branches, the number of pods per plant, the number of grains per plant, and the weight of grains per plant, which plays a significant role in improving soybean yield. Therefore, the present invention provides the following application schemes:

[0010] Application of BN1 gene in improving soybean yield, wherein the BN1 gene is BN1a gene and / or BN1b gene; the sequence of BN1a gene is shown in SEQ ID NO.1; the sequence of BN1b gene is shown in SEQ ID NO.3.

[0011] The method of increasing soybean yield includes increasing the number of pods per plant, the number of grains per plant and the weight of grains per plant.

[0012] Specifically, the amino acid sequence of the protein encoded by the BN1a gene is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the BN1b gene is shown in SEQ ID NO.4.

[0013] The application of the above-mentioned BN1 gene in increasing the number of soybean branches.

[0014] The above-mentioned BN1 gene is used as a target for increasing soybean yield.

[0015] The above-mentioned BN1 gene is used as a target for increasing the number of soybean branches.

[0016] The above-mentioned BN1 gene is used as a target in constructing soybean strains with improved yield.

[0017] The above-mentioned BN1 gene is used as a target in constructing soybean lines with increased branch number.

[0018] Application of the inhibitor of the BN1 gene or its protein in improving soybean yield and / or increasing the number of soybean branches.

[0019] The use of the expression inhibitor of the BN1 gene in constructing soybean lines with improved yield and / or increased branch number, wherein the expression inhibitor includes gene silencing, knockdown and knockout agents.

[0020] Application of the BN1 gene fragment shown in SEQ ID NO.5 as a target for constructing soybean lines with increased yield and / or increased branch number.

[0021] In addition, the present invention also provides a method for breeding multi-branched high-yield soybean varieties, the method comprising silencing, knocking down or knocking out the BN1 gene of soybean; the BN1 gene is the BN1a gene and / or the BN1b gene; the sequence of the BN1a gene is shown in SEQ ID NO.1, and the sequence of the BN1b gene is shown in SEQ ID NO.3.

[0022] As an alternative embodiment, CRISPR / Cas9 technology is used to knock out the above-mentioned editing sites.

[0023] The present invention has the following beneficial effects:

[0024] The present invention obtains soybean bn1a-bn1b double mutants by CRISPR / Cas9 method. The study found that the soybean bn1a-bn1b double mutants have increased branch number, increased pod number per plant, increased grain number per plant, and increased grain weight per plant. Soybeans are improved by using BN1a gene and BN1b gene to create multi-branched high-yield soybean germplasm resources, thereby increasing soybean yield, which has good application prospects and value in soybean breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Editing pattern of soybean bn1a-bn1b double mutant.

[0026] Figure 2 Field phenotypic identification of soybean bn1a-bn1b double mutant plants (Figure A shows the phenotypic diagram of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure B shows the plant height measurement results of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure C shows the main stem node number measurement results of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure D shows the main stem node number measurement results of soybean bn1a-bn1b double mutant plants and control W82 plants Figure 1 shows the results of branch number determination; Figure E shows the results of pod number per plant of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure F shows the results of grain number per plant of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure G shows the results of grain weight per plant of soybean bn1a-bn1b double mutant plants and control W82 plants; Figure H shows the results of 100-grain weight of soybean bn1a-bn1b double mutant plants and control W82 plants). DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] Experimental methods of CRISPR / Cas9 technology refer to "Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015 Aug; 8(8): 1274-84."

[0030] Experimental methods of soybean whole-plant transgenic technology refer to "Chen L, Nan H, Kong L, Yue L, Yang H, Zhao Q, Fang C, Li H, Cheng Q, Lu S, Kong F, Liu B, Dong L. Soybean AP1 homologs control flowering time and plant height. J Integr Plant Biol. 2020 Dec; 62 (12): 1868-1879."

[0031] Example 1 Creation of soybean bn1a-bn1b knockout mutant

[0032] The sequence of soybean BN1a gene is (5'-3'):

[0033]

[0034] The amino acid sequence of the protein encoded by the soybean BN1a gene is:

[0035] MDKSEQTQQQQQQQQHVMGVAAGASQMAYSSHYPTASMVASGTPAVTAPSPTQAPAAFSSSAHQLAYQQAQHFHHQQQHQQQLQMFWSNQMQEIEQTIDFKNHSLPLARIKKIMKADEDVRMISAEAPVIFAKAC EMFILELTLRSWIHTEENKRRTLQKNDIAAAISRNDVFDFLVDIIPRDELKEEGLGITKATIPLVGSPADMPYYYVPPQHPVVGPPGMIMGKPIGAEQATLYSTQQPRPPVAFMPWPHTQPLQQQPPQHQQTDS(SEQ ID NO.2).

[0036] The sequence of soybean BN1b gene is (5'-3'):

[0037]

[0038] The amino acid sequence of the protein encoded by the soybean BN1b gene is:

[0039] MDKSEQTQQQHQHGMGVATGASQMAYSSHYPTAPMVASGTPAVAVPSPTQAPAAFSSSAHQLAYQQAQHFHHQQQQHQQQLQMFWSNQMQEIEQTIDFKNHSLPLARIKKIMKADEDVRMISAEAPVIFAKACEM FILELTLRSWIHTEENKRRTLQKNDIAAAISRNDVFDFLVDIIPRDELKEEGLGITKATIPLVNSPADMPYYYVPPQHPVVGPPGMIMGKPVGAEQATLYSTQQPRPPMAFMPWPHTQPQQQQPPQHQQTDS(SEQ ID NO.4).

[0040] A DNA sequence of 22 deoxyribonucleotides was selected as the knockout editing site of soybean BN1a and BN1b genes, and the sequence was (5'-3'): CCAAATGGCCTATTCTTCTCAC (SEQ ID NO. 5).

[0041] The exact location of this site in the soybean genome is at bases 2308-2329 on the BN1a gene on chromosome 08, and the chromosome coordinates are Chr08:1308493-138514.

[0042] At the same time, this site is also located at the 2012-2033 bases on the BN1b gene on chromosome 15 of the soybean genome, and the chromosome coordinates are Chr15:49384513-49384534.

[0043] Using soybean W82 as the background, the BN1a and BN1b genes of soybean (variety W82) were knocked out simultaneously by CRISPR / Cas9 technology and soybean whole-plant transgenic technology, and a homozygous soybean bn1a-bn1b double mutant was obtained. The editing method of the bn1a-bn1b double mutant is as follows Figure 1 As shown, the results showed that Bn1a deleted 374 bp at the target site and Bn1b deleted 386 bp at the target site.

[0044] Example 2 Identification of branch number and single plant yield phenotype of bn1a-bn1b double mutant and control W82

[0045] The bn1a-bn1b double mutant and the control W82 were planted in the fields in Shijiazhuang for phenotypic identification. The plant height, number of main stem nodes, number of branches, number of pods per plant, number of grains per plant, grain weight per plant, and 100-grain weight of the double mutant and the control W82 were measured respectively.

[0046] The phenotype, plant height, number of main stem nodes, and number of branches of soybean bn1a-bn1b double mutant plants and control W82 plants were measured as follows: Figure 2 As shown in Figures A, B, C, and D, the results showed that there was no significant difference in plant height between the soybean bn1a-bn1b double mutant plants and the control, but the number of branches was significantly increased compared with the control.

[0047] In addition, the results of the number of pods per plant, number of grains per plant, grain weight per plant, and 100-grain weight of the soybean bn1a-bn1b double mutant plants and the control W82 plants were as follows: Figure 2 As shown in Figures E, F, G, and H, the results showed that there was no significant difference in 100-grain weight between the soybean bn1a-bn1b double mutant plants and the control, but the results of the number of pods per plant, the number of grains per plant, and the grain weight per plant were significantly better than those of the control.

[0048] The number of pods, grains and grain weight per plant are the main traits that determine the yield of legume crops, which proves that the mutations of BN1a and BN1b genes have the potential to increase soybean yield.

[0049] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The application of BN1 gene in increasing soybean yield is characterized by: The BN1 gene is the BN1a gene and / or the BN1b gene; the sequence of the BN1a gene is shown in SEQ ID NO.1; the sequence of the BN1b gene is shown in SEQ ID NO.

3.

2. Use of the BN1 gene described in claim 1 in increasing the number of soybean branches.

3. Use of the BN1 gene described in claim 1 as a target for increasing soybean yield.

4. Use of the BN1 gene of claim 1 as a target for increasing the number of soybean branches.

5. Use of the BN1 gene described in claim 1 as a target in constructing a soybean strain with improved yield.

6. Use of the BN1 gene as claimed in claim 1 as a target in constructing a soybean line with increased branch number.

7. Use of the inhibitor of the BN1 gene or its protein according to claim 1 in increasing soybean yield and / or the number of soybean branches.

8. Use of the BN1 gene expression inhibitor of claim 1 in constructing a soybean strain with improved yield and / or increased branch number, characterized in that: The expression inhibitors include gene silencing, knockdown, and knockout agents.

9. Application of the BN1 gene fragment shown in SEQ ID NO.5 as a target for constructing soybean lines with increased yield and / or increased branch number.

10. A method for cultivating a multi-branched high-yield soybean variety, characterized in that: Silencing, knocking down or knocking out the BN1 gene of soybean; the BN1 gene is the BN1a gene and / or the BN1b gene; the sequence of the BN1a gene is shown in SEQ ID NO.1, and the sequence of the BN1b gene is shown in SEQ ID NO.3.

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