A high protein yield and its sequence in soybean

Abstract

The present application relates to the field of biotechnology, in particular to a kind of high-yield protein and its sequence in soybean application.The present application provides the application of protein MBS1 in the regulation of soybean yield.The present application shows that protein MBS1 is enhanced in soybean by transgenic experiment and field experiment, and realizes the significant high-efficiency yield increase in field.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of a high-yield protein and its sequence in soybeans. Background Technology

[0002] Studies have shown that overexpression of the monocot-specific gene OsDREB1C in rice can increase yield by 41–68%, but only by 17–22% in wheat. Overexpression of yield factors such as GY3, OsPIL11, and MOC1 often has no effect or is even harmful in soybeans and rapeseed. Other studies have shown that while overexpression of the VPZ (Violaxanthin de-epoxidase, PsbS, Zeaxanthin epoxidase) gene in potatoes accelerates the induction and relaxation of non-photochemical quenching (NPQ), it can negatively impact yield under certain conditions. Transferring NbSTOMR across species into rice does not improve rice resistance to rice false smut and rice blast. This is the first experimental evidence demonstrating that PRR also exhibits a similar "restricted taxonomic function" (RTF) phenomenon to NLR during cross-species transfer. Furthermore, studies have shown that ectopic expression of TaBG1 increases wheat grain size and alters its nutritional characteristics, but does not lead to increased yield. In rice: previous studies have shown that overexpression of OsBG1 in rice increases grain size and simultaneously improves yield. Other crop validation: applying rice research results to wheat, increasing TaBG1-A expression does indeed lead to larger grains, but the number of grains per plant decreases, resulting in no significant increase in total yield, limiting its ability to increase yield alone. Therefore, the introduction of high-yield genes does not necessarily yield good results.

[0003] Soybean (Glycine max (L.) Merr.) is an annual herbaceous plant belonging to the genus Glycine in the family Leguminosae. As a major food and economic crop in my country, its yield plays a vital role in the country's food security. Summary of the Invention

[0004] In view of this, the present invention provides a high-yield protein and its sequence for use in soybeans.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Firstly, this invention provides overexpression MBS1 The application of genes in increasing soybean yield, the MBS1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0006] Secondly, the present invention also provides a method for increasing soybean yield or a method for cultivating transgenic soybeans that increase soybean yield, comprising: overexpression MBS1 Gene; The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0007] Thirdly, the present invention also provides a high-yield soybean with an inserted conversion agent; The transformant carries MBS1 Gene; The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0008] This study found that in soybeans, lines overexpressing MBS1 showed a significant increase in yield during field trials. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0010] Figure 1 Soybeans MBS1 Schematic diagram of gene structure; Figure 2 Show MBS1 A map of gene transformation vectors (vectors into which the MBS gene is inserted). Figure 3 The diagram shows PCR detection of transgenic soybeans that tested positive. W "-" represents the non-transgenic wild-type (W) control, and "+" represents the vector plasmid positive control. MBS1#1-MBS1#8 represent eight transgenic lines from generation T0, all of which tested positive in this study. Lane M represents the molecular weight marker, with marker sizes of 15kb, 10kb, 7.5kb, 5kb, 2.5kb, 2kb, 1kb, 0.75kb, and 0.5kb, respectively. Figure 4 Test strips detected genetically modified soybeans as positive; Figure 5 show MBS1 Soybean phenotype; Figure 6 Show MBS1 Yield per genetically modified soybean plant; data are expressed as mean ± standard deviation, using Student's... t -test performs statistical analysis. express P <0.001. Detailed Implementation

[0011] This invention discloses a high-yield protein and its sequence in soybeans. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0012] Use Student's t -test performs statistical analysis.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0014] The present invention will be further illustrated below with reference to the embodiments: Example 1 MBS1 Application in improving soybean yield—Preparation of transgenic soybean plants Application of soybean homologous genes MBS1 Enhanced expression improves soybean yield. Specifically: 1. Preparation of expression vector pGMCQ1 (MBS1) soybeans MBS1 The gDNA fragment of the gene (LOC100784149, GLYMA_09G250600v4) in soybean (1.9 kb, containing MBS1 The gene (including its promoter and terminator) was double-digested with BamHI and XbaI and ligated between the corresponding restriction sites on the pGMCQ1 transformation vector backbone to obtain the recombinant vector; in this vector... MBS1 Expression is driven by its own promoter; the marker gene is the Bar gene (glufosinate / Basta resistance, derived from Streptomyces hygroscopicus), driven by the 35S promoter and terminated by NOS.

[0015] The sequence of the MBS1 protein, 105 amino acids (as shown in SEQ ID No. 1). MTGKAKPKKHTAKEIAAKVDAATTNRGGGKAGMKDRTGLEKGGHAKYECPHCKVTAPDVKSMQIHHDARHPKIPFEEDKVVNLHATTSVPESSKPRPGVRGSLKK- The nucleic acid sequence encoding MBS1 (GLYMA_09G250600v4) is 318 bp (as shown in SEQ ID No. 2). atgacagggaaggcgaagccgaagaagcacggcgaaggagatcgcggcgaaggtggacgcggcgaccacgaaccgcggcggcgggaaggcgggtatgaaggaccgaaccgggttggagaagggcgggcacgcgaaatacgagtgccctcactgcaag gtgacggcgccggacgtgaaatcgatgcagatccaccacgacgcgcgtcaccccaagatccccttcgaggaggacaaagttgtcaatcttcacgccaccaccagcgttcccgagtcctccaagcctcgccccggtgttcgcggaagcctcaagaagtga 2. Genetically modified soybean plants 1) Transformation: The recombinant vector pGMCQ1-MBS1 was transformed into Agrobacterium EHA105 and used to transform cotyledonary node explants of the soybean variety Zhonghuang 35. Resistance was selected using glufosinate-ammonia (Basta) as a selection marker, and T0 generation transgenic soybean plants were obtained. A total of 8 independent transgenic lines (MBS1-1, MBS1-2…MBS1-8) were obtained. The specific method is as follows: (1) Seed disinfection: Select robust and mature soybean seeds and use chlorine disinfection method (100 mL of sodium hypochlorite with 8% available chlorine added to 5 mL of concentrated hydrochloric acid to produce chlorine gas) to continuously fumigate and sterilize in a fume hood for 16–20 h; after sterilization, ventilate in a clean bench for about 24 h to dissipate residual chlorine gas, place in a petri dish, seal and store at 4 ℃ for later use.

[0016] (2) Pre-culture (germination): Sterilized seeds are inoculated into germination medium with the hilum facing down and cultured in the dark at 25 ℃ for 1 day.

[0017] (3) Preparation of Agrobacterium bacterial culture: 1 µL of plasmid was added to 50 µL of Agrobacterium competent cells, mixed well, and transferred to an electroporation cuvette. After electroporation, 1 mL of LB liquid medium was added, mixed well, and transferred to a centrifuge tube. The culture was carried out at 30 °C and 180 rpm for 30 min. Agrobacterium (EHA105) carrying the target gene plasmid was spread on the corresponding medium for initial activation. After 48 h of culture, the bacterial cells were collected and activated again on a new medium. After 24 h of culture, the bacterial cells were collected, resuspended in the infection solution, vortexed, and the bacterial culture was adjusted to OD using a spectrophotometer. 600 =0.5 (for backup)

[0018] (4) Infection and co-culture: After the germinated soybean explants are scratched, they are poured into the bacterial solution to complete the infection; the infection solution is discarded, and the explants are placed on the co-culture medium with filter paper, and co-cultured at 25 ℃ in the dark for 3–5 days.

[0019] (5) Recovery culture: Select explants that have grown well and are free from pollution after co-culture, cut off the end of the hypocotyl, insert them into the recovery solid culture medium, and recover culture for 7–10 days.

[0020] (6) Screening culture: Explants with clustered shoots were inoculated into screening medium and cultured under 16 h light / 8 h dark for 21 days.

[0021] (7) Elongation culture: The selected healthy buds were transferred to elongation culture medium and cultured for 21 days under 16 h light / 8 h darkness.

[0022] (8) Rooting culture: When the shoots grow to about 5 cm, they are transferred to the rooting medium for further screening. They are cultured for 21 days with 16 h light and 8 h darkness.

[0023] (9) Hardening off seedlings: Remove the seedlings that have passed the positive test from the culture medium, wash off the culture medium attached to the roots, and transplant them into seedling trays containing nutrient soil. Harden off the seedlings for 3–4 weeks at 27 ℃ with 16 h light / 8 h darkness.

[0024] Screening agent: The medium for screening, elongation and rooting stages contains 4–8 mg / L of glufosinate (Basta).

[0025] 2) Identification: Genomic DNA was extracted from the leaves of regenerated plants, and PCR was performed using insert-specific primers to identify the integration of exogenous genes; glufosinate resistance protein was detected using transgenic PAT / bar colloidal gold test strips.

[0026] Primers used: Primer sequence 5'→3' oNS407TAATCATCGCAAGACCGGCA (as shown in SEQ ID No. 3); oNS408TGTCAATCTTCACGCCACCA (as shown in SEQ ID No. 4); The reaction conditions were: first 94℃ for 2 minutes, then 98℃ for 10 seconds, 60℃ for 30 seconds, 72℃ for 1 minute and 30 seconds, for a total of 35 cycles, and finally 72℃ for 5 minutes. The PCR products were electrophoresed using a 1% agarose gel. Figure 3 All transgenic plants were able to amplify fragments of about 500 bp in size, while non-transgenic plants could not amplify any fragments.

[0027] Colloidal gold test strip detection: The presence of bar / pat proteins in transgenic plants was directly identified using the Bar test strip method. The results are as follows: Figure 4 As shown.

[0028] The colloidal gold test strip results showed that the control line (C line) of all test strips developed normally, indicating that the test was effective. Some samples showed a red band at the T line position, which was judged as positive for the target transgenic protein; samples that did not show a T line were judged as negative.

[0029] MBS1 The overexpression of the gene is deterministic, and since the gene is a functional gene, the overexpression of the MBS1 protein is predictable.

[0030] Example 2 MBS1 Application in improving soybean yield—Determination of yield in transgenic soybean plants The T1 generation homozygous transgenic lines and wild-type Zhonghuang 35 were planted in the field in Shunyi District, Beijing (Shunyi Experimental Base of Chinese Academy of Agricultural Sciences). The yield per plant and yield composition were determined at maturity. change MBS1 Cultivation and management parameters for genetically modified soybeans in field trials: change MBS1 The genetically modified soybean material and wild-type control, Zhonghuang 35, underwent field trials at the Shunyi Experimental Base of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, in Housangyuan Village, Zhaoquanying Town, Shunyi District, Beijing. The experimental site was surrounded by roads, walls, woodlands, and lawns, representing an artificially controlled ecological type, and possessed the isolation and management conditions required for field trials of transgenic materials.

[0031] Test materials include transfer MBS1Genetically modified soybean lines and the wild-type Zhonghuang 35 control were used. Transgenic materials were planted using the transgenic seedling transplanting method, and transplanted and managed according to soybean field trial management practices. Planting specifications were 50 cm row spacing and 25 cm plant spacing, with one seedling per hole. Each material was divided into 3 plots, with 6 rows per plot and 65 plants per row. The transgenic materials and the wild-type control were planted in the same experimental site under the same cultivation and management conditions.

[0032] Fertilizer and water management should follow the conventional field management practices for soybeans in the Beijing area. Apply 1000-1500 kg / mu of high-quality farmyard manure as base fertilizer, or 20 kg / mu of NPK compound fertilizer; for seed fertilizer, apply 10 kg / mu of diammonium phosphate and 5 kg / mu of urea. During the flowering and podding stage, foliar spray with 0.5-0.75 kg / mu of potassium dihydrogen phosphate, 0.5 kg / mu of urea, and micronutrient fertilizers, applied in 2-3 applications. After sowing, irrigate once according to soil moisture; irrigate once each during the flowering and podding stage and the grain-filling stage if drought occurs; maintain soil moisture content at approximately 70%-80% of field capacity in the early grain-filling stage, and drain water promptly after rain.

[0033] Field weed control primarily involves post-sowing weed control, using herbicides such as metolachlor, cypermethrin, pendimethalin, and oxyfluorfen, depending on the weed population. If the weed control is ineffective, post-emergence weeding can be performed at the 4-6 compound leaf stage. Large weeds should be removed manually in the mid-to-late stages. Pest and disease control focuses on prevention and monitoring. For early-stage diseases, foliar sprays with fungicides such as metalaxyl or carbendazim can be applied 2-3 times. For early-stage pests, 10% imidacloprid wettable powder at a 2000-fold dilution and 5% lambda-cyhalothrin emulsifiable concentrate at a 1500-fold dilution can be applied every 7-10 days, for a total of 3-5 applications depending on the severity of the infestation. All treatment plots should use the same herbicide, dosage, and application time for uniform management.

[0034] The experimental site was spatially isolated and managed by designated personnel. After maturity, the genetically modified soybeans were harvested manually and separately to prevent seed scattering and mixing with conventional materials. Harvested genetically modified soybean seeds were individually bagged, numbered, registered, and stored by designated personnel. Remaining soybean stalks, stems, and leaves were disposed of according to the management requirements for the genetically modified experimental base. The results are shown in Table 1.

[0035] Table 1. Soybean Yield and Yield Composition (Transferred from MBS1)

[0036] Measurements were taken from 16 individual plants: like Figure 5 , 6 As shown, compared to the wild type, the conversion... MBS1 Soybean yield per plant increased significantly, with an increase of approximately +50.01%. P <0.001, n>15), indicating enhanced expression in soybean. MBS1 It can significantly increase soybean yield.

[0037] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression MBS1 The application of genes in increasing soybean yield is characterized by, The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

2. A method for increasing soybean yield or a method for cultivating transgenic soybeans that increase soybean yield, characterized in that, include: overexpression MBS1 Gene; The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. A high-yield soybean, characterized in that, Including exogenous insertions MBS1 Gene; The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

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