A high protein yield and its sequence in rape

Abstract

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

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 rapeseed. 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] Rapeseed is a dicotyledonous plant and an important economic crop in my country. Its yield plays a vital role in my 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 rapeseed.

[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 rapeseed 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 rapeseed yield or a method for cultivating transgenic rapeseed that increases rapeseed 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 rapeseed with an insert conversion body; 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 rapeseed, the yield of lines overexpressing MBS1 was significantly increased in planting 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 This shows that the PCR test results for genetically modified rapeseed were positive. Figure 2 The MBS1 transgenic rapeseed phenotype is shown; Figure 3 This shows the yield of a single MBS1 transgenic oilseed 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 applied in rapeseed. 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 rapeseed 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 rapeseed yield—Preparation of transgenic rapeseed plants application MBS1 Enhanced gene expression in rapeseed improves rapeseed yield. Details are as follows: 1. Preparation of expression vector pTCK303 (MBS1) pTCK303 (MBS1): Rice MBS1 The gDNA fragment (0.8 kb) of the gene (Os06g0708600) was digested with BamHI and SacI and ligated to the corresponding restriction sites in the backbone of the transformation vector pTCK303 to obtain the recombinant vector. In this vector, OsMBS1 is expressed by the promoter of the maize Ubiquitin1 gene and terminated by the Nos terminator. The marker gene is HPT (hygromycin phosphotransferase, hygromycin resistance, 35S promoter, NOS terminator).

[0014] The protein has the following amino acid sequence (as shown in SEQ ID No. 1), containing 114 amino acids, with a molecular weight of 11.90 kDa. MTGKAKPKKHTAKEIAAKIDAATTNRGGGKAGQKDRLGQDKGGHAKLECPLCKTTAPDIKSMQIHHEARHPKLPFEPDKLNNLHGGGAGAAAAAGEAAASSSKPKPGVRGSLKK The sequence of the nucleic acid molecule encoding the above protein is shown in SEQ ID No. 2: atgacggggaaggcgaagccgaagaagcacacggcgaaggagatcgcggcgaagatcgacgcggcgacgacgaacaggggcggcgggaaggccgggcagaagggccctggggcaggacaagggcggccacgccaagctcgagtgccctctctgcaagaccaccgcccccg acatcaagtccatgcagatccaccacgaggcgcgccaccccaagctccccttcgagcccgacaagctcaacaacctccacggcggcggcgccggcgccgccgccgccgccggtgaggccgccgcctcctcctccaagcccaagcccggcgtccgcggcagcctcaagaagtaa 2. Genetically modified rapeseed plants 1) Transformation: The recombinant vector was transferred into Agrobacterium GV3101 and used to transform hypocotyl explants of Brassica napus Westar. T0 generation transgenic rapeseed plants were obtained through hygromycin selection. Transgenic seedlings were obtained and identified as transgenic lines. Details are as follows: (1) Seed cleaning and germination: The seeds were disinfected with 75% ethanol for 30–60 s and washed with sterile water once (1 min); treated with 0.15% mercuric chloride for 10 min and washed with sterile water twice (1 min each); then washed with sterile water for 30 min, dried on sterile filter paper and inoculated into germination medium, and cultured in the dark at 23 ℃ for 5–6 days.

[0015] (2) Pre-culture: The hypocotyl of the germinating seedlings was cut into segments of 0.4–0.6 cm as explants, inoculated into pre-culture medium, and cultured under light at 23 ℃ for 2–3 days.

[0016] (3) Agrobacterium infection and co-culture: Agrobacterium (GV3101) was picked and placed in the infection solution to prepare OD. 600 The explants were inoculated into the bacterial suspension at a concentration of 0.2 and incubated for 10 min. After being removed and dried on sterile filter paper, they were inoculated into co-culture medium and incubated in the dark at 23°C for 48–72 h.

[0017] (4) Destermination (delayed screening): The co-cultured explants were inoculated into a destermination medium and cultured at 23 ℃ under light for 6 days.

[0018] (5) Screening / differentiation: The sterilized explants were inoculated into the screening / differentiation medium (about 30 explants per plate), cultured at 23 ℃ under light, and the plates were changed every 15 days.

[0019] (6) Rooting culture: Inoculate the differentiated buds into the rooting medium and culture them under light at 23 ℃ until they root.

[0020] Screening agent: Hygromycin B 20–40 mg / L was added to the culture medium during the screening, differentiation and rooting stages; positive identification was performed by PCR detection using the following exogenous gene detection primers.

[0021] 2) Identification: Genomic DNA was extracted from the leaves of the regenerated plants and PCR was performed using transgene-specific primers to identify the integration of exogenous genes.

[0022] 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).

[0023] If a transgene is inserted, a PCR product of approximately 840 bp can be amplified.

[0024] 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.

[0025] MBS1 The overexpression of the gene is deterministic, and since the gene is a functional gene, the overexpression of the MBS1 protein is predictable.

[0026] Example 2: Application of MBS1 in improving rapeseed yield—Determination of yield of transgenic rapeseed plants change MBS1 Cultivation and management parameters for genetically modified rapeseed planting trials change MBS1 Genetically modified rapeseed materials and wild-type Westar controls were grown in pots in a transgenic experimental greenhouse. The greenhouse was equipped with the isolation and management conditions required for the cultivation of transgenic materials.

[0027] The greenhouse used test materials including... MBS1 Genetically modified rapeseed lines and wild-type Westar controls. After artificial seedling cultivation, rapeseed was transplanted into pots and placed in a greenhouse for further cultivation. One plant was planted per pot, with a pot diameter of 25 cm and a substrate depth of 20 cm. The pots were planted 10 cm apart. The transgenic material and the wild-type control were grown in the same greenhouse under the same cultivation and management conditions.

[0028] In the greenhouse, water and fertilizer management is carried out according to conventional rapeseed cultivation: an appropriate amount of compound fertilizer is added to the cultivation substrate as base fertilizer, and during the growing season, watering is carried out regularly and compound fertilizer is applied with the water to ensure normal plant growth and development. In the greenhouse, watering is carried out in a timely manner according to the substrate moisture condition to maintain a suitable substrate moisture content and avoid waterlogging or drought stress.

[0029] Prevention is the primary approach to pest and disease control in greenhouses. When pests such as aphids, cabbage caterpillars, diamondback moths, sclerotinia rot, and downy mildew occur, appropriate registered pesticides should be selected for timely control. Consistent control measures and management standards should be adopted for all treatments.

[0030] At maturity, each plant is harvested manually to avoid seed scattering; the seeds are threshed, cleaned, and weighed separately, and the yield and yield composition traits of each plant are recorded. Genetically modified rapeseed seeds are sealed in special bags and managed by designated personnel; after harvesting, residual plants, fallen leaves, and threshing residues are uniformly inactivated in accordance with the biosafety management requirements for genetically modified organisms.

[0031] Transgenic lines were planted with wild-type Westar, and yield and yield composition traits such as main stem diameter, number of siliques per plant, and grain weight per plant were measured. The results are shown in Table 1.

[0032] Table 1 (transfer) MBS1 Biomass and yield traits of rapeseed T0 generation

[0033] Results: Compared with the wild type, the T0 generation of transgenic rapeseed showed significantly increased main stem diameter, number of siliques per plant, and grain weight per plant, indicating enhanced expression of transgenic rapeseed. MBS1 It can significantly increase the biomass (stem diameter) and yield (number of pods and grain weight) of rapeseed.

[0034] 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 rapeseed yield is characterized by, The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No.

2.

2. A method for increasing rapeseed yield or a method for cultivating transgenic rapeseed that increases rapeseed 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 rapeseed variety, characterized in that, Including external insertions MBS1 Gene; The MBS1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

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