Application of soybean GmSTP gene in improving soybean seed yield and / or oil content

Abstract

This invention relates to the application of the soybean GmSTP gene in improving soybean seed yield and / or oil content, belonging to the field of plant genetic engineering technology. This invention provides the application of the soybean GmSTP gene in improving soybean seed yield and / or oil content, and the genomic nucleotide sequence of the soybean GmSTP gene is shown in SEQ ID NO.1. This invention clones the soybean GmSTP gene, constructs a recombinant expression vector containing this gene and recombinant Agrobacterium, and creates transgenic soybean plants overexpressing the soybean GmSTP gene. The results of the examples show that the soybean GmSTP gene is a key gene regulating soybean seed yield and oil content. This invention provides genetic resources for the breeding and improvement of high-yield, high-oil soybean varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the soybean GmSTP gene in improving soybean seed yield and / or oil content. Background Technology

[0002] Soybean [Glycine max (L.) Merr.] is an important dual-purpose crop for grain, oil, and feed in my country and the world. However, due to factors such as small planting area and low yield, my country's total soybean production is far below domestic demand, and my country relies heavily on imports (up to 80%), seriously threatening my country's food and oil security. Therefore, using molecular breeding techniques to independently design and cultivate soybean varieties with excellent target traits such as high oil content and high yield is crucial for the development of my country's soybean industry.

[0003] Soybean yield and oil content are both quantitative traits, regulated by a complex network of numerous genes. In recent years, researchers have discovered a large number of chromosomal regions and single nucleotide polymorphisms (SNPs) that affect yield and oil content through quantitative trait locus (QTL) mapping and genome-wide association studies (GWAS). However, the discovery and functional study of major genes with high phenotypic contributions are still relatively limited, which to some extent restricts the application of key major genes in the genetic improvement of high-oil and high-yield soybeans.

[0004] Therefore, by using genetic engineering techniques to identify key genes that regulate soybean yield and oil content and to analyze their genetic regulatory mechanisms, we can provide important theoretical basis and genetic resources for achieving high-oil, high-yield soybean molecular breeding with independent intellectual property rights, in order to address the import dependence problem faced by my country's soybean industry development. Summary of the Invention

[0005] In order to overcome the problems existing in the background art, the present invention provides the application of soybean GmSTP gene in improving soybean seed yield and / or oil content, which can provide usable gene resources for molecular breeding of high-oil and high-yield soybeans.

[0006] The application of the soybean GmSTP gene provided by this invention in improving soybean seed yield and / or oil content is characterized in that the genomic nucleotide sequence of the soybean GmSTP gene is shown in SEQ ID NO.1.

[0007] The application of the soybean GmSTP gene provided by this invention in improving soybean seed yield and / or oil content is characterized in that the nucleotide sequence of the CDS coding frame of the soybean GmSTP gene is shown in SEQ ID NO.2.

[0008] The present invention relates to the application of the protein encoded by the soybean GmSTP gene in improving soybean seed yield and / or oil content, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.3, and the application is to overexpress the protein in soybean plants.

[0009] The present invention provides a recombinant vector for improving soybean seed yield and / or oil content, characterized in that the recombinant vector contains the CDS coding frame nucleotide sequence of the soybean GmSTP gene as shown in SEQ ID NO.2.

[0010] In the above technical solution, the recombinant vector is the pGmUBI3301-GmSTP vector, which is prepared by recombinantly linking the CDS sequence of the GmSTP gene shown in SEQ ID NO.2 into the pGmUBI3300 vector.

[0011] This invention provides a method for increasing soybean seed yield and / or oil content, characterized by comprising the following steps: (1) Cloning the soybean GmSTP gene, wherein the nucleotide sequence of the CDS coding frame of the soybean GmSTP gene is shown in SEQ ID NO.2; (2) The cloned soybean GmSTP gene was constructed into an expression vector to obtain a recombinant vector; (3) The recombinant vector is transformed into Agrobacterium to obtain recombinant Agrobacterium; (4) Soybean explants were infected with the recombinant Agrobacterium and screened to obtain positive soybean plants that overexpressed the soybean GmSTP gene.

[0012] In the above technical solution, the method for constructing the recombinant vector in step (2) includes: using soybean seed cDNA as an amplification template, amplifying the CDS sequence of the GmSTP gene shown in SEQ ID NO.2, and recombinantly linking the CDS sequence into the pGmUBI3300 vector to prepare the pGmUBI3301-GmSTP recombinant vector.

[0013] The present invention provides the application of recombinant Agrobacterium in improving soybean seed yield and / or oil content, characterized in that the recombinant Agrobacterium contains the recombinant vector as described in claim 4 or 5.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention cloned the soybean GmSTP gene, constructed a recombinant expression vector containing this gene and recombinant Agrobacterium, and created transgenic soybean plants overexpressing the soybean GmSTP gene using Agrobacterium-mediated transformation. The yield (grain weight) of individual soybean plants overexpressing the GmSTP gene was measured. Compared with the control variety Williams82 (W82), the seed yield per plant of GmSTP gene-overexpressing plants was significantly increased, the seed oil content was significantly increased, and the seed protein content was decreased. This indicates that the GmSTP gene is a key functional gene regulating soybean seed yield and oil content. This invention utilizes this gene for soybean germplasm improvement, enabling the targeted breeding of high-yielding, high-oil-content superior soybean lines. It provides valuable genetic resources and novel breeding targets for the selection of high-quality, high-yielding soybean varieties, germplasm resource innovation, and molecular-assisted breeding, possessing significant scientific research value and broad prospects for industrial application. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the pGmUBI3300-GmSTP transgenic vector in an embodiment of the present invention; Figure 2 This is an identification diagram of the herbicide test strip in an embodiment of the present invention; Figure 3 This is a semi-quantitative detection map of STP expression in soybean leaves in an embodiment of the present invention; Figure 4 This is a graph showing the gene expression levels of transgenic positive plants in an embodiment of the present invention; Figure 5 This is a diagram showing the yield of a single transgenic plant in an embodiment of the present invention; Figure 6 This is a diagram showing the oil and protein content of the seeds of the transgenic plants in this embodiment of the invention. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0018] Where the following description relates to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0019] In the description of this invention, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of these terms in this invention based on the specific circumstances. Furthermore, in the description of this invention, unless otherwise stated, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] This invention uses the soybean variety Williams82 (WT / Wm82) as the genetic transformation recipient material, hereinafter referred to as WT or Wm82. The nucleotide sequence of the soybean GmSTP gene (Glycine max Wm82.a2.v1 version gene number: Glyma.08G059700) in this invention is shown in SEQ ID NO.1; its CDS coding sequence is 1560 bp in length, and its nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0022] SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: MPAVGGISNGGGKEYPGSLTPFVTVTCIVAAMGGLIFGYDIGISGGVTSMDPFLLKFFPSVFRKKNSDKTVNQYCQYDSQTLTMFTSSLYLAALLSSLVASTVTRRFGRKLSMLFGGLLFLVGALINGFA QHVWMLIVGRILLGFGIGFANQSVPLYLSEMAPYKYRGALNIGFQLSITVGILVANVLNYFFAKIHGGWGWRLSLGGAMVPALIITIGSLVLPDTPNSMIERGDREKAKAQLRRVRGIDDVEEEFNDLVA ASESSRKVEHPWRNLLQRKYRPHLTMAVLIPFFQQLTGINVIMFYAPVLFSSIGFKDDSALMSAVITGVVNVVATCVSIYGVDKWGRRALFLEGGVQMVICQAVVAAAIGAKFGIDGNPGDLPKWYAVVV VLFICIYVSAFAWSWGPLGWLVPSEIFPLEIRSAAQSINVSVNMFFTFLIAQVFLTMLCHMKFGLFIFFAFFVLIMTFFIYFFLPETKGIPIEEMNQVWKAHPFWSRFVENDDYGNGVEMGRGTAIKEV.

[0023] Example 1: Cloning of the soybean GmSTP gene 1. Seeds of wild-type soybean Wm82 were collected 25 days after flowering, and total RNA was extracted using TransZol Plant reagents. Using the seed RNA as a template, cDNA was obtained by reverse transcription using the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for PCR kit. RNA extraction and cDNA reverse transcription were performed according to the manufacturer's instructions.

[0024] 2. Using cDNA as a template, PCR amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.) and primers GmSTPcds-F and GmSTPcds-R. The nucleotide sequence of primer GmSTPcds-F is shown in SEQ ID NO.4, and the nucleotide sequence of primer GmSTPcds-R is shown in SEQ ID NO.5. The PCR program used was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec; 56℃ annealing for 30 sec; 72℃ extension for 2 min; 32 cycles of 72℃ extension for 5 min.

[0025] SEQ ID NO.4: ATGCCTGCGGTAGGAGGTATTAG SEQ ID NO.5: CTACACTTCTTTGATAGCAGTTCCCC 3. The PCR amplification products were separated by agarose gel electrophoresis. After the target fragment was recovered by gel excision, TA cloning was performed using the 5 min TA / Blunt-Zero Cloning Kit (Novizan), and then transformed into Top10 competent cells.

[0026] 4. Perform colony PCR identification, pick positive colonies, shake them to extract plasmids, and then send them to the company for sequencing to obtain the cloning vector GmSTP-TOPO containing the GmSTP gene.

[0027] Example 2 Construction of a recombinant vector overexpressing the soybean GmSTP gene 1. Using the SacI restriction site and its upstream sequence, and the BamHI restriction site and its downstream sequence of pGmUBI3300 as homologous arms, primers GmSTP-GmUBI3300-F and GmSTP-GmUBI3300-R were designed. The nucleotide sequence of primer GmSTP-GmUBI3300-F is shown in SEQ ID NO. 6, and the nucleotide sequence of primer GmSTP-GmUBI3300-R is shown in SEQ ID NO. 7. PCR amplification was performed using the GmSTP-TOPO plasmid as a template. After agarose gel electrophoresis and gel recovery of the target fragment, the GmSTP fragment with the homologous arm of the pGmUBI3300 vector was obtained.

[0028] SEQ ID NO.6: ttgttgactcgacaggagctcATGCCTGCGGTAGGAGGTATT SEQ ID NO.7: atgatctttgtaatcggatccCACTTCTTTGATAGCAGTTCCCC 2. The pGmUBI3300 vector was double-digested with restriction endonucleases SacI and BamHI. The undigested plasmid was used as a control for electrophoresis detection. The linearized plasmid was then recovered by gel electrophoresis.

[0029] 3. Using the ClonExpress® II One Step Cloning Kit, take an appropriate amount of the gene and vector fragments obtained in steps 1 and 2 according to the instructions, prepare a homologous recombination reaction system, react at 37°C for 30 min, and then transform the reaction product into E. coli.

[0030] 4. Colony PCR identification was performed. Positive colonies were picked, cultured, and plasmids were extracted. These were then sent to the company for sequencing, ultimately yielding the overexpression transgenic vector pGmUBI3301-GmSTP containing the GmSTP gene. The vector structure is shown below. Figure 1 As shown.

[0031] Example 3 Preparation of recombinant bacteria overexpressing the soybean GmSTP gene 100 μL of Agrobacterium EHA105 competent cells (Beijing Cooler Master Technology Co., Ltd.) were taken from a -80℃ freezer. An appropriate amount of the pGmUBI3301-GmSTP vector plasmid constructed in Example 2 was added. The cells were incubated on ice for 15 min, flash-frozen in liquid nitrogen for 5 min, transferred to a 37℃ constant temperature water bath for 5 min of heat shock, and finally placed on ice for 5 min of recovery. 500 μL of antibiotic-free YEP liquid medium was added, and the cells were incubated at 28℃ with a shaker at 220 rpm for 2.5 h. 100 μL of the bacterial culture was spread onto YEP solid medium containing 50 mg / L rifampicin and 50 mg / L kanamycin, and cultured at 28℃ for 2 days to obtain Agrobacterium carrying the pGmUBI3301-GmSTP expression vector, named EHA105 / pGmUBI3301-GmSTP.

[0032] Example 4: Obtaining transgenic soybean plants overexpressing the soybean GmSTP gene 1. Select plump, smooth, and mold-free Wm82 soybean seeds for surface disinfection, then place them on MS basal medium and incubate in the dark at 25℃ for 3-5 days to obtain uncontaminated cotyledonary nodes as explants.

[0033] 2. Activate Agrobacterium EHA105 / pGmUBI3301-GmSTP containing the vector to be transformed. When the OD600=0.5, immerse the explants in Agrobacterium and infect for 3 minutes, stirring repeatedly during the process. After co-culture, induction culture and differentiation culture, rooting culture is carried out to finally obtain transgenic plants.

[0034] 3. Collect a small number of leaves from T0 generation transgenic plants and use the Bar test strip method to detect whether the plants express Bar / pat protein. Continue to plant positive plants and record the seeds after harvest as T1 generation seeds.

[0035] 4. After sowing the T1 generation seeds, test whether they are positive plants using the same method as in step 3. If they are positive plants, continue planting until the T2 generation seeds are harvested.

[0036] 5. T2 generation seeds were sown, and Bar test strips were used to detect the expression of GmSTP in the leaves. RNA was extracted from leaves of positive plants and cDNA was obtained by reverse transcription. Primers GmSTP-RT-F and GmSTP-RT-R were designed, with the nucleotide sequence of GmSTP-RT-F shown in SEQ ID NO.7 and the nucleotide sequence of GmSTP-RT-R shown in SEQ ID NO.8. RT-PCR experiments were performed using cDNA from the leaves of positive plants as templates to detect whether GmSTP expression was higher than that of Wm82. Positive plants with expression levels higher than Wm82 were designated as GmSTP-OE plants.

[0037] SEQ ID NO.7: CAGAAAAGTGGAGCACCCGT SEQ ID NO.8:ACCCGATGGAGCTGAACAAC The results are as follows Figure 2 As shown, two horizontal lines on the Bar test strip represent transgenic plants GmSTP-OE1 and GmSTP-OE2 expressing the Bar protein, indicating transgenic positive plants; while Wm82 has only one horizontal line, indicating a transgenic negative plant. The RT-PCR results are as follows... Figure 3 As shown, GmSTP-OE1 and GmSTP-OE2 were expressed at higher levels than Wm82 in the same PCR cycle.

[0038] 6. Seeds from individual plants of Wm82, GmSTP-OE1, and GmSTP-OE2 during the T3 generation development were collected. Seeds from two stages, approximately 30 days and 50 days after flowering, were collected and designated as S3 and S4. These were then sent to the company for transcriptome sequencing to detect the gene expression profiles of the seeds at different stages.

[0039] The results are as follows Figure 4As shown, in the two positive plants GmSTP-OE1 and GmSTP-OE2, the GmSTP gene expression was significantly higher than that of Wm82 in both S3 and S4 stages, indicating that GmSTP-OE1 and GmSTP-OE2 are two soybean plants with GmSTP gene overexpression.

[0040] Example 5: Seed trait evaluation of transgenic plants overexpressing the soybean GmSTP gene The number and weight of seeds, oil content, and protein content of individual plants of the two overexpressing plants, GmSTP-OE1 and GmSTP-OE2, were determined (e.g., Figure 5 and Figure 6 (As shown in the figure). The results showed that compared with Wm82, the number and weight of seeds per plant were significantly increased in GmSTP-OE1 and GmSTP-OE2 plants overexpressing the GmSTP gene, and the oil content was significantly increased, while the protein content was decreased.

[0041] The above results indicate that overexpression of the GmSTP gene can promote an increase in the number of seeds per soybean plant and improve the seed oil content, suggesting that the GmSTP gene is a key gene regulating soybean yield, oil content, and protein content, providing a new genetic resource for the genetic improvement of soybeans.

[0042] In the accompanying drawings of this embodiment, the same or similar reference numerals correspond to the same or similar components. In the description of this application, it should be understood that if terms such as "upper," "lower," "left," "right," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting this application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0043] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The use of soybean GmSTP gene in improving soybean seed yield and / or oil content, characterized in that, The genomic nucleotide sequence of the soybean GmSTP gene is shown in SEQ ID NO.

1.

2. The application of the soybean GmSTP gene in improving soybean seed yield and / or oil content, characterized in that, The CDS coding frame nucleotide sequence of the soybean GmSTP gene is shown in SEQ ID NO.

2.

3. The application of the protein encoded by the soybean GmSTP gene in improving soybean seed yield and / or oil content, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.3, and the application is to overexpress the protein in soybean plants.

4. A recombinant carrier for increasing soybean seed yield and / or oil content, characterized in that, The recombinant vector contains the CDS coding frame nucleotide sequence of the soybean GmSTP gene as shown in SEQ ID NO.

2.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is the pGmUBI3301-GmSTP vector, which is prepared by recombinantly linking the CDS sequence of the GmSTP gene shown in SEQ ID NO.2 into the pGmUBI3300 vector.

6. A method for increasing soybean seed yield and / or oil content, characterized in that, Includes the following steps: (1) Cloning the soybean GmSTP gene, wherein the nucleotide sequence of the CDS coding frame of the soybean GmSTP gene is shown in SEQ ID NO.2; (2) The cloned soybean GmSTP gene was constructed into an expression vector to obtain a recombinant vector; (3) The recombinant vector is transformed into Agrobacterium to obtain recombinant Agrobacterium; (4) Soybean explants were infected with the recombinant Agrobacterium and screened to obtain positive soybean plants that overexpressed the soybean GmSTP gene.

7. The method for increasing soybean seed yield and / or oil content according to claim 6, characterized in that, The method for constructing the recombinant vector in step (2) includes: using soybean seed cDNA as an amplification template, amplifying the CDS sequence of the GmSTP gene shown in SEQ ID NO.2, and recombinantly linking the CDS sequence into the pGmUBI3300 vector to prepare the pGmUBI3301-GmSTP recombinant vector.

8. The application of a recombinant Agrobacterium in improving soybean seed yield and / or oil content, characterized in that, The recombinant Agrobacterium contains the recombinant vector as described in claim 4 or 5.

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