A high protein yield and its sequence in corn

Abstract

The present application relates to the field of biotechnology, in particular to a kind of high-yield protein and its sequence in corn application.The present application provides the application of protein MBS1 in the regulation of corn biomass.The present application shows that protein MBS1 can be expressed in corn by transgenic experiment and greenhouse experiment, and can realize the significant and efficient increase of corn biomass.

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 maize. 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 PRRs also exhibit a similar "restricted taxonomic function" (RTF) phenomenon to NLRs 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] Maize is a tall, annual herbaceous plant belonging to the genus *Zea* of the Poaceae family. It is one of the three major staple food crops and an important silage crop. As a major food and feed crop in my country, its yield and biomass play a vital role in the country's food security. Summary of the Invention

[0004] In view of this, the present invention provides an application of a high-yield protein and its sequence in maize.

[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 maize biomass, 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 maize biomass or a method for cultivating transgenic maize that increases maize biomass, 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 maize 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 maize, lines overexpressing MBS1 showed a significant increase in biomass during greenhouse cultivation. 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 The image shows transgenic positive maize detected by PCR. "+" indicates a positive control for the vector plasmid, and "-" indicates a non-transgenic wild-type (W) control. MBS1#29-MBS1#31 represent two 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 2 Test strips detected positive results for genetically modified corn; Figure 3 This shows the MBS1 transgenic maize phenotype; Figure 4 Show MBS1 Biomass per transgenic maize plant; data are expressed as mean ± standard deviation, using Student's data. t -test performs statistical analysis. express P <0.001. Detailed Implementation

[0011] This invention discloses a high-yield protein and its sequence in maize. 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] The raw materials and reagents used in the application of the high-yield protein and its sequence in maize provided by this invention are all commercially available.

[0013] The present invention will be further illustrated below with reference to the embodiments: Example 1 MBS1 Application in improving maize biomass—Preparation of transgenic maize plants application MBS1 Homologous genes are enhanced in maize, improving maize biomass and yield. Details are as follows: 1. Preparation of expression vector pGMCQ1 (MBS1) The promoter of the soybean GmMBS gene was constructed by adding the rice OsMBS1 gene (containing 5'UTR, CDS, and 3'UTR), and resistance screening was performed using the bar gene (glufosinate resistance).

[0014] The insertion fragment was inserted into the pGMCQ1 vector via the BamHI and XbaI sites.

[0015] The promoter of the soybean MBS1 (Glyma.09G250600) gene is shown in SEQ ID No. 5: The inserted rice OsMBS1 gene (773 bp) has a coding region (CDS) marked with bold underline, flanked by 5' UTR and 3' UTR, as shown in SEQ ID No. 6: atcaaacacaaatagggttagcgagaatcggcggcggcggcggcggcggcggcgagagcggagagagacaggagggggcaaccgcggcggcggcggcggcggcgggggcgag atgacggggaaggcgaagcc gaagaagcacacggcgaaggagatcgcggcgaagatcgacgcggcgacgacgaacaggggcggcgggaaggccggg cagaaggaccggctggggcaggacaagggcggccacgccaagctcgagtgccctctctgcaagaccaccgcccccg acatcaagtccatgcagatccaccacgaggcgcgccaccccaagctccccttcgagcccgacaagctcaacaacct ccacggcggcggcgccggcgccgccgccgccgccggtgaggccgccgcctcctcctccaagcccaagcccggcgtc cgcggcagcctcaagaagtaa ccgcaatcgaatccatcatcacccgaatctgctactactaccgcccgccgccgctcgctccggtgtgcttacttgctgctgcttgctgttttgaattattactactgtgtcatgatgtgccgtgtgatagatgcgttgattcgcgcacatatacatatgt gaccgtgttccgtcgcgtcttcgtcggcatgttttagctatgatgtttagtttatgtgtcactactctatgatgctgtcaatggtgtcaatggatgataatgtttatgtctgtcactgctcaaacaattgctaaatctgagtaaagaaaaaggtgatggc The amino acid sequence of MBS1, the protein used to transform maize, is shown in SEQ ID No. 1. It contains 114 amino acids and has a molecular weight of 11.90 kDa. MTGKAKPKKHTAKEIAAKIDAATTNRGGGKAGQKDRLGQDKGGHAKLECPLCKTTAPDIKSMQIHHEARHPKLPFEPDKLNNLHGGGAGAAAAAGEAAASSSKPKPGVRGSLKK The sequence (345 bp) of the nucleic acid molecule encoding the aforementioned protein MBS1 is shown in SEQ ID No. 2: atgacggggaaggcgaagccgaagaagcacacggcgaaggagatcgcggcgaagatcgacgcggcgacgacgaacaggggcggcgggaaggccgggcagaagggccctggggcaggacaagggcggccacgccaagctcgagtgccctctctgcaagaccaccgcccccg acatcaagtccatgcagatccaccacgaggcgcgccaccccaagctccccttcgagcccgacaagctcaacaacctccacggcggcggcgccggcgccgccgccgccgccggtgaggccgccgcctcctcctccaagcccaagcccggcgtccgcggcagcctcaagaagtaa A maize transformation vector was constructed using the plant binary expression vector pGmCQ1 as its backbone. This vector contains the left and right boundary sequences of T-DNA, retains the NOS-bar-PolyA selection marker expression cassette within the T-DNA, and includes the backbone elements required for vector replication and bacterial selection. A promoter fragment was designed based on the promoter sequence of the soybean GmMBS1 gene (Glyma.09G250600). A fragment from the rice OsMBS1 gene was also used; this fragment contains the 5′UTR, complete CDS, and 3′UTR of OsMBS1, with the CDS being 345 bp and encoding 114 amino acids.

[0016] The modified soybean GmMBS1 promoter fragment was ligated to the rice OsMBS1 gene fragment in the sequence "GmMBS1 promoter-OsMBS1 5′UTR-OsMBS1 CDS-OsMBS1 3′UTR" to obtain the PGm-OsMBS expression fragment. BamHI and XbaI restriction sites were artificially synthesized and introduced at both ends of this expression fragment. The pGmCQ1 vector and the PGm-OsMBS expression fragment were double-digested with BamHI and XbaI, respectively, and then recovered. The PGm-OsMBS fragment was inserted into the original target gene expression cassette of the pGmCQ1 vector, placing it after the original upstream regulatory sequence and before the NOSterminator, thus obtaining the maize transformation vector.

[0017] 2. Genetically modified maize plants 1) Transformation: The recombinant vector pGMCQ1 (MBS1) was transformed into Agrobacterium EHA105, which was then used to transform the embryogenic embryos of the maize inbred line B104. Resistance selection was performed using glufosinate-ammonia (Basta) as a selection marker, and T0 generation transgenic maize plants were obtained. Transgenic lines MBS1#29 and MBS1#31 were obtained. Specific methods: (1) Sample collection, disinfection and embryo collection: Select corn embryos about 10–14 days after pollination and about 1.0–1.5 mm in length as recipients; after removing the outer shell of the ear, disinfect (soak in 70% ethanol for about 30 s → soak in 20% sodium hypochlorite solution for 15–20 min → rinse with sterile water 3–5 times), peel off the embryos, inoculate them with the scutellum facing up on the induction medium, and culture them in the dark for 2–3 days to keep them in an active division state.

[0018] (2) Preparation of Agrobacterium bacterial culture: A single colony of Agrobacterium (EHA105) containing the recombinant plasmid was picked and inoculated into liquid LB containing the corresponding antibiotic, and cultured at 28 ℃ with shaking until OD. 600 =0.5–0.8, centrifuge to collect bacterial cells, resuspend in infection medium containing acetylsyringone (AS, which induces T-DNA transfer), and adjust to OD. 600 ≈0.2 (0.2–0.5).

[0019] (3) Infection and co-culture: Immerse the pretreated embryos in Agrobacterium bacterial solution for 10–20 min (shaking gently during the process); remove and aspirate excess bacterial solution, and transfer the embryos along with the bacterial solution to a co-culture medium containing AS, and incubate in the dark at 22–25 ℃ for 2–5 days.

[0020] (4) Debacterial removal and screening: After co-culture, the embryos were washed 3–4 times with liquid medium containing cephalosporin or carbenicillin to remove residual Agrobacterium; the embryogenic callus was inoculated on induction medium and cultured in the dark at 28 ℃ for 7–10 days, then transferred to screening medium and cultured in the dark at 28 ℃ for 2 weeks. The callus that survived the first screening was used for the second screening.

[0021] (5) Differentiation and rooting: The selected resistant embryogenic callus was inoculated on predifferentiation medium and cultured in the dark at 28 ℃ for about 10 days, and then transdifferentiated on 25 ℃ light culture (16 h light / 8 h dark) until differentiation and seedling emergence; when the shoots grew to 2–3 cm, they were trans-rooted on rooting medium and cultured on 25 ℃ light culture until the root system was fully developed, and after hardening, they were transplanted to the greenhouse substrate.

[0022] Screening agent: The culture medium for screening, differentiation and rooting stages contains 10–40 mg / L of glufosinate (Basta).

[0023] 2) Identification: Genomic DNA was extracted from the leaves of regenerated plants using the CTAB method, 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.

[0024] Exogenous gene integration detection Molecular detection was performed on transgenic plants using specific primers located on the OsMBS1 insertion fragment and the NOS terminator to detect the integration of the foreign gene. The primers were: oNS264:5'aagccgaagaagcacacgg (as shown in SEQ ID No. 3); 1460-NosR: 5' tgtataattgcgggactctaatc (as shown in SEQ ID No. 4).

[0025] If a transgene is inserted, a PCR product of approximately 840 bp can be amplified.

[0026] 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 1 All transgenic plants were able to amplify a fragment of about 840 bp, while non-transgenic plants could not amplify any fragment.

[0027] Colloidal gold test strip detection: The Bar test strip method is used to directly detect whether transgenic plants contain Bar / PAT (glufosinate resistance) protein to help identify positive lines.

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

[0029] Overexpression of the MBS1 gene is certain, and since this gene is a functional gene, the overexpression of the MBS1 protein is predictable.

[0030] Example 2: Application of MBS1 in improving maize biomass—Determination of biomass in transgenic maize plants change MBS1 Parameters for greenhouse cultivation and management of genetically modified corn: change MBS1 Genetically modified maize materials and wild-type control B104 were grown in pots in a transgenic experimental greenhouse. The greenhouse was equipped with temperature and light control facilities and isolation management for transgenic materials.

[0031] Test materials include transfer MBS1 Genetically modified maize lines and the wild-type B104 control. Transgenic maize seedlings were transplanted into cultivation pots filled with nutrient substrate, one plant per pot. The pot diameter was approximately 30 cm, the substrate depth was approximately 25 cm, and the pots were spaced approximately 50 cm apart. They were then cultivated in a greenhouse with a day / night temperature of approximately 28 ℃ / 22 ℃ and a photoperiod of approximately 14 h light / 10 h dark. The transgenic material and the wild-type control were grown in the same greenhouse under the same cultivation and management conditions.

[0032] In the greenhouse, water and fertilizer management is carried out according to conventional corn cultivation: an appropriate amount of compound fertilizer is added to the cultivation substrate as base fertilizer, and urea and other fertilizers are applied with water during key growth periods such as jointing and silking to ensure normal plant growth and development; water is applied in a timely manner according to the substrate moisture to avoid drought or waterlogging.

[0033] In greenhouses, pest and disease control is primarily preventative. Regular surveys are conducted from the seedling stage to maturity. When thrips, aphids, fall armyworm, corn borer, or leaf spot occur, appropriate registered pesticides are used for timely control. All treatments are treated uniformly using the same pesticide, dosage, and application time. At maturity, individual plants are harvested, and aboveground biomass (plant height, stem diameter, aboveground dry weight) and other traits are measured. Harvested transgenic maize material and seeds are individually bagged, numbered, registered, and stored by designated personnel. Residual plants, cobs, and kernels are uniformly inactivated according to transgenic biosafety management requirements to prevent seed scattering and material mixing.

[0034] Measurement: The transgenic lines were planted with wild-type B104, and the aboveground biomass (plant height, stem diameter, and aboveground dry weight) was measured. The results are shown in Table 1.

[0035] Table 1 (converted to MBS1) Maize Biomass

[0036] As shown in the table, compared with the wild-type control B104, the two independent transgenic lines MBS1#29 and MBS1#31, which enhanced the expression of MBS1 in maize, showed improved aboveground biomass-related traits. The plant height, stem diameter, and single-plant biomass (aboveground dry weight) of wild-type B104 were 173.26 cm, 2.28 cm, and 63.39 g, respectively; the corresponding values ​​for transgenic line MBS1#29 were 186.5 cm, 2.37 cm, and 87.82 g, and for MBS1#31 were 185.39 cm, 2.63 cm, and 92.55 g.

[0037] Based on the average values ​​of two independent transgenic lines, compared to the wild-type, transgenic maize showed an increase of approximately 7.32% in plant height, 9.65% in stem diameter, and 42.27% in biomass per plant (above-ground dry weight). The increase in biomass per plant was the largest, and both independent transgenic lines consistently showed higher values ​​than the wild-type control. (Based on Student's...) t The test results were highly significant (P<0.001); plant height and stem diameter also showed an increasing trend. The increase in plant height, stem diameter and single plant dry weight corroborated each other, which together constituted the overall increase in aboveground biomass of transgenic maize.

[0038] The above results indicate that enhanced expression of MBS1 in maize can significantly increase aboveground biomass. It should be noted that aboveground biomass is an important agronomic trait for silage (feed) maize and other crops where vegetative growth is the primary harvesting factor. Therefore, the protein MBS1 has clear application value in improving aboveground biomass in maize, and provides experimental evidence for its use in maize biomass improvement.

[0039] 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 maize biomass is characterized by, The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

2. A method for increasing maize biomass or a method for cultivating transgenic maize that increases maize biomass, 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 corn variety, 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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