Application of GmDRP1 gene and its encoded protein in regulating soybean reproductive growth period and yield

Abstract

The present invention discloses GmDRP1 The application of genes and encoded proteins in regulating soybean reproductive growth and yield. GmDRP1 Gene expression in soybean varieties can prolong the reproductive growth period and significantly increase the yield of soybeans. GmDRP1 Gene editing to obtain homozygous gene-edited mutants can shorten the reproductive growth period of soybeans and significantly reduce yields, indicating that the present invention GmDRP1 Genes play an important role in the length of soybean reproductive growth period and yield. The present invention provides important gene resources for soybean molecular breeding and also provides an effective means for high-yield soybean.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and more specifically, to GmDRP1 Application of genes and encoded proteins in regulating soybean reproductive growth period and yield. Background Art

[0002] Soybean is an important cash crop, cultivated worldwide and a major source of edible oil and plant protein. The soybean growth cycle is divided into two phases: the vegetative phase and the reproductive phase. Studies have shown that the length of the reproductive phase is significantly correlated with soybean yield, and appropriately extending the reproductive phase can significantly increase soybean yield. While several major QTLs and genes have been mapped for soybean growth traits, few specific QTLs and genes for the reproductive phase have been reported. Therefore, identifying the major genes controlling the reproductive phase is crucial for breeding high-yield soybeans.

[0003] GmDRP1 belongs to the NAC family of transcription factors, one of the largest transcription factor families in plants. NAC transcription factors are composed of the initials NAM (No apical meristem in petunia), ATAF (Arabidopsis transcription activation factor), and CUC (cup-shaped cotyledon). This family of transcription factors comprises a complex, plant-specific superfamily that is widespread across species. NAC transcription factor family members share high homology in their N-termini, a domain consisting of approximately 150 amino acids. Studies have shown that NAC family members play a role in various physiological and biochemical processes in plants, including shoot apical meristem formation, lateral root formation, flower development, cell division, cell wall development, leaf senescence, and secondary wall formation. They are also involved in responses to a variety of biotic and abiotic stresses. However, in soybean, no NAC family members have been reported to be involved in the length of the reproductive growth period or yield. Therefore, studying the regulatory mechanism of soybean NAC family members on the length of soybean reproductive growth period and yield is of great significance, and also provides new genes and materials for improving soybean yield. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide GmDRP1 The application of genes and encoded proteins in regulating the length of soybean reproductive growth period and yield will GmDRP1 Gene expression in soybean varieties can extend the reproductive growth period of soybeans and increase soybean yield; mutation of the soybean GmDRP1 gene can shorten the reproductive growth period of soybeans and lead to reduced yield.

[0005] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0006] soybeans GmDRP1 The gene is located on soybean chromosome 5, has a reading frame length of 897 bp, and encodes 298 amino acids. GmDRP1 The plant expression vector of the gene was transformed into soybean, such as Huachun 6. Compared with the control material Huachun 6, the transgenic plants obtained by overexpression showed the phenotype of extended reproductive growth period and increased yield. GmDRP1 Gene editing was performed and homozygous plants were obtained. It was found that the reproductive growth period of homozygous plants was shortened and the yield was reduced. GmDRP1 Genes are positively regulating soybean reproductive growth period and yield.

[0007] Therefore, the present invention provides GmDRP1 The following new uses of genes:

[0008] GmDRP1 The application of the gene in any of the following: (1) regulating the length of the reproductive growth period and yield of soybean; (2) preparing a product for regulating the length of the reproductive growth period and yield of soybean; GmDRP1 The gene is a gene encoding any one of the following proteins A1) to A3):

[0009] A1) a protein with the amino acid sequence shown in SEQ ID No. 2;

[0010] A2) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID No. 2, which has more than 90% identity with the protein of A1) and is associated with the length of the reproductive growth period and yield of soybean;

[0011] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0012] GmDRP1 Application of a gene-related biological material in any of the following: (1) application in regulating the length of the reproductive growth period and yield of soybean; (2) application in preparing a product for regulating the length of the reproductive growth period and yield of soybean; the related biological material is any one of the following B1) to B9):

[0013] B1) a nucleic acid molecule encoding any one of the proteins A1) to A3) above;

[0014] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0016] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0017] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0018] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0019] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0020] B8) a nucleic acid molecule that reduces the expression of any of the proteins in A1) to A3) above;

[0021] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0022] The present invention also provides GmDRP1 The use of gene-related biological materials in any of the following: (1) application in extending the reproductive growth period of soybean and increasing soybean yield; (2) application in preparing products that extend the reproductive growth period of soybean and increase soybean yield; 3) application in cultivating soybean varieties with extended reproductive growth period and increased yield; the related biological materials are any one of B1) to B7) above.

[0023] Furthermore, B1) the nucleic acid molecule is a cDNA molecule or a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 1.

[0024] The present invention also provides a method for regulating the length of the reproductive growth period and yield of soybeans, by increasing the GmDRP1 or the expression level of its encoded protein, to obtain soybeans with prolonged reproductive growth period and increased yield;

[0025] Or, using genetic engineering to GmDRP1 The gene or its promoter is modified so that GmDRP1 The gene function is missing or weakened, or the expression level is reduced, thereby obtaining soybeans with shortened reproductive growth period and reduced yield;

[0026] described GmDRP1 The gene is a gene encoding any one of the following proteins A1) to A3):

[0027] A1) a protein with the amino acid sequence shown in SEQ ID No. 2;

[0028] A2) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID No. 2, which has more than 90% identity with the protein of A1) and is associated with the length of the reproductive growth period and yield of soybean;

[0029] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0030] Furthermore, the method of increasing the GmDRP1 Or the method for increasing the expression level of the protein encoded by it is to overexpress the protein shown in SEQ ID No. 2 in soybean; or introduce the gene encoding the protein shown in SEQ ID No. 2 into soybean.

[0031] Specifically, any vector for directing exogenous gene expression in plants is used to GmDRP1 Genes introduced into soybean plant cells can produce soybean plants with extended reproductive growth period and increased yield. GmDRP1 When recombining plant expression vectors, any enhanced promoter or constitutive promoter is added before its transcription start nucleotide, such as the cauliflower mosaic virus 35S promoter and the ubiquitin promoter of corn. They can be used alone or in combination with other plant promoters. In addition, in order to facilitate the screening of transgenic plants or cells, all plant expression vectors can be processed to add enzyme genes or luminescent compound genes (luciferase genes, GUS genes, etc.) that can produce color changes when expressed in plants, antibiotic resistance markers (spectinomycin, kanamycin, etc.), or chemical resistance marker genes (glyphosate resistance genes, herbicide resistance genes, etc.).

[0032] Furthermore, the GmDRP1 The method of gene function loss or weakening, or expression level reduction is to GmDRP1 The gene is the target, an sgRNA sequence based on CRISPR-Cas9 is designed, a DNA fragment containing the encoding sgRNA sequence is connected to a vector carrying CRISPR-Cas9, and soybeans are transformed to obtain transgenic soybeans with missing or weakened gene function or reduced expression level.

[0033] Furthermore, the action site of the sgRNA is located at GmDRP1 within the gene coding region.

[0034] Preferably, the nucleotide sequence of the sgRNA action site is 5'-GGAGAACTCCAATTACCAC-3'.

[0035] Preferably, the transformation is mediated by Agrobacterium.

[0036] The present invention also provides the use of transgenic soybeans obtained by any of the above methods in plant breeding. The breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides GmDRP1 The application of genes and encoded proteins in regulating the length of soybean reproductive growth period and yield. GmDRP1 Gene expression in soybean varieties can prolong the reproductive growth period and significantly increase the yield of soybeans. GmDRP1 Gene editing to obtain homozygous gene-edited mutants can shorten the reproductive growth period and significantly reduce soybean yield, indicating that GmDRP1 The gene positively regulates the reproductive growth period and yield of soybeans, and plays an important role in the length of the reproductive growth period and yield of soybeans. The present invention provides an important gene resource for soybean molecular breeding and also provides an effective means for high-yield soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The gene that controls the reproductive growth period and yield of soybean in Example 1 GmDRP1 Among them, Figure 1 A is the Manhattan plot of all chromosomes in the genome-wide association study (GWAS) of soybean yield traits; B is the Manhattan plot of chromosome 5 in the genome-wide association study (GWAS) of soybean yield traits; C is the QTL positioning map of the population generated by Guizao 1 and Vencedora; D is GmDRP1 Expression profiles in soybean varieties Guizao 1 and Vencedora.

[0040] Figure 2 Schematic diagram of mutation and expression of gene editing materials and overexpression materials in Example 2. Figure 2 A is a schematic diagram of the mutation of two mutants obtained in the Huachun 6 background; B is a schematic diagram of the mutation of two overexpression materials obtained in the Huachun 6 background GmDRP1 Expression analysis diagram; C is two overexpression materials obtained in Young background GmDRP1 Expression analysis diagram.

[0041] Figure 3 For transgenic materials and mutant phenotype analysis. Figure 3A is a schematic diagram of the material phenotypes of mutants and overexpression lines obtained in the Huachun 6 background; B is a statistical diagram of the soybean reproductive growth period phenotypes of mutants and overexpression lines obtained in the Huachun 6 background; C is a statistical diagram of the soybean yield phenotypes of mutants and overexpression lines obtained in the Huachun 6 background; D is a schematic diagram of the material phenotypes of the overexpression lines obtained in the Young background; E is a statistical diagram of the soybean reproductive growth period phenotypes of the overexpression lines obtained in the Young background; F is a statistical diagram of the soybean yield phenotypes of the overexpression lines obtained in the Young background. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0044] The soybean material Vencedora is disclosed in the literature (Prado, RDM, Franco, CF, & Puga, AP (2010). Macronutrient deficiencies in soybean cv. brsmg 68 (vencedora)cultivated in nutritive solution. Comunicata Scientiae, 1) and is a soybean variety; Guizao 1 and Huachun 6 are disclosed in the literature (Zhong Caixia, Zhong Kaizhen, Zhao Yunyun, Chen Lin, Nian Hai, Ma Qibin, Yang Cunyi. Evaluation of phosphorus efficiency of Brazilian soybean resources and their derivative varieties in South China [J]. Chinese Journal of Oil Crops, 2013, 35(02): 162-170.) and are soybean varieties; Young is disclosed in the literature (Abdel-Haleem H, Jr T, Rufty TW, et al.Quantitative trait loci controlling aluminum tolerance in soybean: Candidategene and single nucleotide polymorphism marker discovery [J]. Molecular Breeding, 2013, 33(4):851-862.) is disclosed as a soybean variety; the above materials are available to the public from the College of Agriculture of South China Agricultural University.

[0045] Example 1 Soybean GmDRP1Gene cloning

[0046] The inventors of the present invention isolated and cloned the NAC transcription factor gene from soybean GmDRP1 The nucleotide sequence is shown in SEQ ID No. 1, and the encoded protein is named GmDRP1 protein. The amino acid sequence is shown in SEQ ID No. 2. The specific cloning method is as follows:

[0047] like Figure 1 As shown, GWAS and map-based cloning were performed by combining genotype and soybean reproductive growth period length and yield phenotype data ( Figure 1 AC), combined with expression profiles, genotype information and homologous gene functional annotations ( Figure 1 (D) Using molecular biology and comparative genomics, we cloned the genes that control soybean reproductive growth and yield. GmDRP1.

[0048] Total RNA was extracted from the leaves of soybean material Vencedora using a plant total RNA extraction kit (TR02, GeneMark), and its integrity was verified by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions of the PrimeScript™ RT reagent Kit with gDNAEraser. PCR amplification was performed using the cDNA as a template and primers SEQ ID No. 3 and SEQ ID No. 4 (SEQ ID No. 3: 5'-GGCTAGGTTCGAAGTTTGGAGGAC-3'; SEQ ID No. 4: 5'-GATGAAACCCTTACATTTTCATTTC-3'). The PCR reaction solution (50 μL) was prepared as follows: 2× Phanta Max Buffer (25 μL), ddH2O (19 μL), dNTP Mix (1 μL), primer F of SEQ ID No. 3 (2 μL), primer R of SEQ ID No. 4 (2 μL), cDNA (1 μL), and Phanta Max Super-Fidelity DNA Polymerase (1 μL). The amplification program was as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 56°C for 15 seconds, and extension at 72°C for 1 minute, for a total of 35 cycles; then, complete extension at 72°C for 5 minutes; and storage at 4°C.

[0049] After amplification, the PCR product was purified and recovered using a common DNA product purification kit (DP204, Tiangen), and then ligated to a pLB vector (pLB Zero Background Quick Ligation Kit, Tiangen). The product was transformed into Escherichia coli TOP10, and a single colony was picked for shake sequencing. The sequencing results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID No. 1, which encoded the protein GmDRP1 as shown in SEQ ID No. 2. The DNA shown in SEQ ID No. 1 was named GmDRP1 Gene.

[0050] Example 2 GmDRP1 Obtaining genetically modified soybeans and gene-edited mutants

[0051] 1. Construction of vector

[0052] Construction of plant expression vector: replace the vector pTF101 (BioVector NTCC Type Culture Collection) with the sequence of SEQ ID No.1 XB Site and SacI The sequence between the sites and other sequences remained unchanged to obtain the recombinant vector pTF101- GmDRP1.

[0053] Construction of gene editing vector: GmDRP1 The target gene sequence in the vector pGES201 (Bai M, Yuan J, Kuang H, et al. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 insoya bean. Plant Biotechnol J. 2020;18(3):721-731. doi:10.1111 / pbi.13239) was replaced with the target gene site (SEQ ID No.5: 5'-GGAGAACTCCAATTACCAC-3') to obtain the recombinant vector pGES201- GmDRP1.

[0054] 2. Use Bio-Rad electroporation instrument to transform the recombinant vector pTF101- GmDRP1 and pGES201- GmDRP1The cells were transformed into Agrobacterium tumefaciens EHA105 (BioVector NTCC Inc.) and then transformed into soybean cotyledonary nodes using the Agrobacterium EHA105-mediated transformation method (Li et al. Optimization of Agrobacterium-Mediated Transformation in Soybean. Front Plant Sci. 2017;8:246. Published 2017 Feb 24. doi:10.3389 / fpls.2017.00246). The culture medium used was the same as that in the reference, as follows: Seed sterilization: Uniform soybean seeds were selected and sterilized with chlorine gas for 12 hours. The chlorine gas was removed in a laminar flow hood and then stored in a refrigerator at 4°C until use. Cotyledonary node infection: Streak EHA105 containing the recombinant plasmid onto YEP solid medium supplemented with spectinomycin (50 mg / L). After incubation at 28°C for 36 hours, single colonies were selected and cultured in YEP liquid medium supplemented with spectinomycin (50 mg / L). Shake overnight at 220 rpm and 28°C, and then the bacterial infection medium was prepared. Sterilized seeds were imbibed in sterile water for 12 hours. The cotyledonary nodes were then scarified using a scalpel in a laminar flow hood. After scarification, the scarified cotyledonary nodes were inoculated in the bacterial infection medium for 1 hour. Co-cultivation: Infected cotyledonary nodes were incubated on co-cultivation medium (CCM) for 5 days until the cotyledons turned green. Bud induction: Cotyledons were transferred to bud induction medium (SI) for 15 days. After incubation, SI was replaced with SI for another 15 days, and clusters of shoots were visible. Shoot Elongation: After two co-cultivation cycles, cotyledon explants with clustered buds are cultured on bud elongation medium SE for a 15-day culture cycle. Rooting: When buds reach approximately 5 cm, they are transferred to rooting medium and cultured until roots are established. The culture is then transferred to soil and cultured until soybean seeds are harvested. Transgenic Line Identification: Harvested transgenic seeds are sown in soil. When the first three leaves are formed, glufosinate-ammonium is applied to confirm positive transgenic lines.

[0055] The present invention obtained two independent transgenic lines in Huachun No. 6 soybean background and Young soybean background (transferred into the recombinant vector pTF101- GmDRP1 ) and two gene-edited mutants (transformed into the recombinant vector pGES201- GmDRP1 ).like Figure 2 As shown in the figure, the two gene editing mutants obtained in the Huachun 6 background were named HKO1 and HKO2; HKO1 and HKO2 deleted 4 (TTAC deletion) and inserted 1 (C insertion) base in the target sequence, respectively ( Figure 2Middle A); Two overexpression lines obtained in the Huachun 6 soybean background were named HOE1 and HOE2, and Huachun 6 was named HWT; GmDRP1 The expression of HOE1 and HOE2 was significantly higher than that in wild-type HWT ( Figure 2 Middle B); Two overexpression lines obtained in the Young background were named YOE1 and YOE2, and Young was named YWT; GmDRP1 The expression levels of YOE1 and YOE2 were significantly higher than those in wild-type YWT ( Figure 2 Middle C).

[0056] Example 3 Genes controlling soybean reproductive growth and yield GmDRP1 Functional verification

[0057] The four independent transgenic lines and two gene-edited mutants prepared in Example 2, as well as the receptor controls Huachun 6 and Young, were used to conduct phenotypic investigations throughout their growth period at Qilin North Farm of South China Agricultural University in Guangzhou, Guangdong Province.

[0058] The overexpression materials (HOE1 and HOE2) and gene editing mutants (HKO1 and HKO2) obtained in the Huachun 6 background showed that the gene editing mutants (HKO1 and HKO2) showed early maturity compared with the recipient Huachun 6 (HWT), while the overexpression materials (HOE1 and HOE2) showed late maturity compared with the recipient Huachun 6 (HWT). Figure 3 Middle A). Statistics of soybean reproductive period length (DRP) showed that the reproductive period length of gene-edited mutants (HKO1 and HKO2) was significantly shorter than that of recipient Huachun 6 (HWT), while the reproductive period length of overexpression materials (HOE1 and HOE2) was significantly longer than that of recipient Huachun 6 (HWT) ( Figure 3 Middle B); Statistics of post-harvest yields revealed that the yields of gene-edited mutants (HKO1 and HKO2) were significantly lower than those of the recipient Huachun 6 (HWT), while the yields of overexpression materials (HOE1 and HOE2) were significantly higher than those of the recipient Huachun 6 (HWT) ( Figure 3 Middle C).

[0059] The overexpression materials (YOE1 and YOE2) obtained in the Young background showed that the overexpression materials (YOE1 and YOE2) showed late maturation compared with the recipient Young (YWT). Figure 3 Middle D). Statistics of soybean reproductive period length (DRP) showed that the reproductive period length of overexpression materials (YOE1 and YOE2) was significantly longer than that of recipient Young (YWT) ( Figure 3 Middle E); Statistics of the yield after harvest showed that the yield of overexpression materials (YOE1 and YOE2) was significantly higher than that of the recipient Young (YWT) ( Figure 3 Middle F).

[0060] The above results show that overexpression in soybean GmDRP1 It can prolong the reproductive growth period of soybean and increase soybean yield, mutation GmDRP1 It shortens the reproductive growth period of soybean and reduces soybean yield, that is, soybean GmDRP1 Genes are positively regulating soybean reproductive growth period and yield.

[0061] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Overexpression GmDRP1 The application of the gene in any of the following: (1) application in increasing soybean yield; (2) application in preparing a product that increases soybean yield; GmDRP1 The gene encodes the protein shown in SEQ ID No.

2.

2. Overexpression GmDRP1 Application of a gene-related biological material in any of the following: (1) application in increasing soybean yield; (2) application in preparing a product that increases soybean yield; the related biological material is any one of the following B1) to B7): B1) a nucleic acid molecule encoding the protein shown in SEQ ID No. 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2); B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

3. The application according to claim 2, characterized in that: B1) The nucleic acid molecule is a cDNA molecule or a DNA molecule whose nucleotide sequence is shown in SEQ ID No.

1.

4. A method for increasing soybean yield, characterized in that: By increasing the target soybean GmDRP1 The expression level of the gene or its encoded protein is increased to obtain soybean with increased yield; GmDRP1 The gene encodes a protein whose amino acid sequence is shown in SEQ ID No.

2.

5. The method according to claim 4, characterized in that: By increasing the GmDRP1 The method for increasing the expression level of the gene or its encoded protein is to overexpress the protein shown in SEQ ID No. 2 in soybean; or to introduce the gene encoding the protein shown in SEQ ID No. 2 into soybean.

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