Rapeseed Seed Oil Content Gene BnSME1 and Its Application

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a gene for regulating the oil content of rapeseed seeds and its application. The gene for regulating the oil content of rapeseed seeds of the present invention is BnSME1 After overexpressing the gene in rapeseed, the oil content of the seeds in the transgenic rapeseed lines increased compared to the control, with the highest increase in oil content reaching 16.26%. This indicates that the gene described in this invention provides a new genetic resource for breeding high-oil rapeseed varieties and can be applied to increase rapeseed oil yield.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a gene BnSME1 for regulating the oil content of rapeseed seeds and an application thereof. Background Art

[0002] Plant oils and fats are an indispensable component of the human diet, important industrial raw materials, and bioenergy sources, playing a crucial role in national economic and social development. In recent years, with people's pursuit of healthier diets, plant oils and fats have gradually replaced animal oils and fats, becoming the primary consumer oil in my country's edible oil market. Rapeseed is a major cash oil crop in my country, with rapeseed oil accounting for 40% of the country's edible oil production. As a winter crop, rapeseed does not compete with grain crops like rice for time and land. Furthermore, rapeseed can be used for edible rapeseed, sightseeing, tourism, beekeeping, and green manure, making it a highly promising cash oil crop. However, rapeseed varieties in my country have low oil content, low yields, and inconsistent quality. Therefore, increasing rapeseed oil yield is a crucial measure to ensure my country's edible oil supply. Increasing rapeseed oil yield per unit area can be achieved by increasing both yield and oil content. However, current research indicates that oil content contributes more significantly to rapeseed oil production, with a 1% increase in rapeseed seed oil content equating to a 2.5% increase in yield. Therefore, increasing the oil content of rapeseed seeds plays an important role in increasing the oil production of rapeseed in my country and maintaining national grain and oil security.

[0003] Current research predicts that rapeseed seed oil content can reach as high as 75%, but the average oil content of existing rapeseed varieties in my country is currently low, leaving significant room for improvement. Rapeseed oil content is a quantitative trait controlled by multiple micro-effect genes and significantly influenced by environmental conditions, making it extremely difficult to significantly increase rapeseed seed oil content using traditional breeding techniques. Currently, with the continuous development and maturity of molecular biology and sequencing technologies, the discovery, isolation, cloning, and functional research of genes regulating rapeseed seed oil content are becoming increasingly in-depth. The use of genetic engineering and gene editing techniques to create new rapeseed varieties with high oil content has become a new direction in rapeseed breeding, with broad development and application prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a gene BnSME1 for regulating the oil content of rapeseed seeds and its application, and to increase the oil content of seeds by increasing the expression level of the gene in rapeseed, thereby achieving the purpose of increasing the oil yield of rapeseed.

[0005] The present invention provides a gene BnSME1 for regulating the oil content of rapeseed seeds. The coding sequence of the BnSME1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0006] The present invention also provides an amplification primer pair for the above gene, comprising BnSME1-F and BnSME1-R. The nucleotide sequence of BnSME1-F is shown in SEQ ID NO.3, and the nucleotide sequence of BnSME1-R is shown in SEQ ID NO.4.

[0007] The present invention also provides a plant expression vector containing the gene BnSME1. Preferably, the base vector of the plant expression vector comprises pCAMBIA1301, and the gene is inserted between BglII and PmlI of the pCAMBIA1301 vector.

[0008] The present invention also provides application of the gene BnSME1 or a plant expression vector thereof in the innovation of rapeseed germplasm resources.

[0009] The present invention also provides application of the gene BnSME1 or a plant expression vector thereof in increasing the oil content of rapeseed seeds.

[0010] The present invention also provides a method for increasing the oil content of rapeseed seeds, which involves overexpressing the BnSME1 gene in rapeseed. The coding sequence of the gene is shown in SEQ ID NO.1.

[0011] Compared with the prior art, the present invention has the following beneficial effects: Through gene isolation and cloning, expression vector construction, and rapeseed transformation, the present invention ultimately obtains the gene BnSME1, which can regulate plant seed oil content. The BnSME1 gene has been applied to crop breeding. Examples have shown that overexpressing the BnSME1 gene in rapeseed increased seed oil content in different transgenic rapeseed lines compared to controls (non-transgenic plants), with the highest increase in oil content in transgenic rapeseed seeds reaching 16.26%. This indicates that the gene described in the present invention provides a new genetic resource for high-oil rapeseed breeding and can be used to increase rapeseed oil yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the construction of recombinant plasmid pCAMBIA1301-BnSME1.

[0013] Figure 2 This is a molecular identification diagram of T1 generation BnSME1 transgenic rapeseed positive seedlings.

[0014] Figure 3 This is the result of oil content determination of BnSME1 transgenic rapeseed seeds. DETAILED DESCRIPTION

[0015] The present invention provides a gene, BnSME1, for regulating the oil content of rapeseed seeds. The nucleotide sequence of gene BnSME1 is shown in SEQ ID NO. 1. To further illustrate the present invention, the gene BnSME1 for regulating the oil content of rapeseed seeds and its applications are described in detail below with reference to the accompanying drawings and examples. Although the above examples provide a detailed description of the present invention, they are merely some, not all, embodiments of the present invention. Those skilled in the art may also derive other embodiments based on this embodiment without inventive step, and such embodiments shall fall within the scope of protection of the present invention.

[0016] In the following examples of the present invention, the experimental material used was Brassica napus L. Zhongshuang 6 rapeseed was bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, and Escherichia coli DH5α competent cells and Agrobacterium GV3101 were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0017] Example 1 Cloning of rapeseed oil content regulatory gene BnSME1 and construction of plant overexpression vector

[0018] Match the backbone vector, design recombination primers BnSME1-F (SEQ ID NO.3: 5'-actcttgaccatggtagatctatggcgagcaccaaagttca-3') and BnSME1-R (SEQ ID NO.4: 5'-gtcacctgtaattcacacgtgttactttcccgtgttcatca-3') at the recombination sites on both ends of the restriction site for PCR. The PCR reaction system in 50 μL preferably includes: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, BnSME1-F 2 μL, BnSME1-R 2 μL, Zhongshuang No. 6 rapeseed cDNA 1 μL and ddH2O 20 μL. The PCR amplification procedure of the present invention preferably includes: 98°C pre-denaturation for 30 seconds; 94°C denaturation for 10 seconds, 65°C annealing for 5 seconds, 72°C extension for 30 seconds, 35 cycles; 72°C extension for 1 minute; and finally storage at 4°C. The amplified PCR product was purified using a PCR product purification kit and stored for future use. The plant overexpression vector pCAMBIA1301 was double-digested with BglII and PmlI restriction endonucleases, detected by 1% agarose gel electrophoresis, and then recovered and stored using a gel recovery kit.

[0019] The PCR-purified product of the BnSME1 gene and the recovered restriction-digested plasmid were recombined using a ligase-independent single-fragment rapid cloning kit. The reaction system consisted of 4 μL of 5× CE II Buffer, 2 μL of Exnase II, 50–200 ng of the linearized vector, and 10–200 ng of the insert. The volume was then supplemented with ddH₂O to a final volume of 20 μL. The recombinant product was transformed into competent E. coli DH5α cells and cultured in an inverted position at 37°C for 12–16 h. Single colonies that emerged were detected by PCR using the upstream primer for the vector's 35S promoter (SEQ ID NO. 5: 5'-acccacgaggagcatcgtgg-3') and the downstream primer for the terminator NOS (SEQ ID NO. 6: 5'-gataatcatcgcaagaccgg-3'). Positive clones confirmed by PCR were sent to Wuhan Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0020] The sequencing results showed that the present invention obtained the full-length CDS of the BnSME1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The expression vector plasmid of the positive single colony with correct sequencing was extracted, and the plasmid was the recombinant plasmid pCAMBIA1301-BnSME1 (the vector construction is shown in Figure 1 shown).

[0021] Example 2 Agrobacterium transformation of pCAMBIA1301-BnSME1 recombinant vector

[0022] 1. Agrobacterium Transformation

[0023] (1) Thaw the competent cells of Agrobacterium tumefaciens GV3101 stored at -80°C to an ice-water mixture at room temperature and place them on ice.

[0024] (2) Use a pipette to draw about 100 ng of the pCAMBIA1301-BnSME1 recombinant plasmid and add it to the GV3101 competent cells. Place them on ice for 5 min, in liquid nitrogen for 5 min, at 37°C for 5 min, and on ice for 5 min.

[0025] (3) Then, add approximately 700 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) and incubate at 28°C in a shaker at 200 rpm for 2–3 h.

[0026] (4) The bacterial solution was spread on LB solid medium supplemented with 50 mg / L kanamycin (Kan), 50 mg / L gentamicin (Gent), and 50 mg / L rifampicin (Rif), and cultured in an inverted incubator at 28°C for 36–48 h.

[0027] (5) PCR detection was performed on the grown Agrobacterium single clone using the upstream primer of the plant expression vector 35S promoter and the downstream primer of the terminator NOS. The positive single clone was shaken to OD 600 =1.8-2.0, and then preserve the bacteria in 50% glycerol and store in -80°C ultra-low temperature freezer for later use.

[0028] 2. Preparation of Agrobacterium Suspension Carrying the BnSME1 Target Gene

[0029] (1) Inoculate the Agrobacterium carrying the target gene in 200 mL of LB liquid medium (containing 50 mg / L Kan, 50 mg / L Gent, and 50 mg / L Rif) at a volume ratio of 1:100, and culture in a shaking incubator at 28°C and 200 rpm until the OD 600 The value is about 0.6;

[0030] (2) Place the bacterial solution in a high-speed centrifuge and centrifuge at 8000 rpm for 15 min. Discard the supernatant and collect the bacterial cells.

[0031] (4) Resuspend the Agrobacterium cells in a resuspension solution (5% sucrose and 0.02% surfactant Silwet L-77) to an OD of 600 The concentration is about 1.0, which is used to prepare Agrobacterium suspension for subsequent rapeseed transformation.

[0032] Example 3 Transformation and Identification Screening of Rapeseed Overexpressing BnSME1

[0033] 1. Transformation of rapeseed using the cotyledon petiole infection method. The specific steps are as follows:

[0034] (1) Zhongshuang No. 6 rapeseed (a public variety, bred by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences) seeds were soaked in 70% ethanol solution for 1 minute, then soaked in mercuric chloride (HgCl2) solution for 13-15 minutes, washed with sterile water 5 times, and then spread on MS culture medium and grown in an artificial climate chamber to obtain sterile rapeseed seedlings for use;

[0035] (2) Take the cotyledon petioles of sterile seedlings of 4-5 days old and place them in the Agrobacterium suspension carrying the pCAMBIA1301-BnSME1 vector for 5-8 minutes, gently shake them, then pour off the bacterial suspension and remove the residual bacterial suspension on the explants; place the transformed cotyledon petioles on a co-culture medium (containing 0.2 mg / L 6-benzyladenine (6-BA), 1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 200 μM acetosyringone) and culture them for 2-3 days;

[0036] (3) The co-cultured explants were transferred to a differentiation medium containing penicillin (Car) and sterilized and differentiated in a dark greenhouse for 5 to 7 days; then, the explants were transferred to a selection medium supplemented with Kan (containing 3 mg / L 6-BA, 0.1 mg / L α-naphthylacetic acid (NAA), 5 mg / L silver nitrate (AgNO3), 400 mg / L Car, and 15 mg / L Kan) for selection, and regenerated green shoots were differentiated from the explants;

[0037] (4) When the regenerated buds grew to 1 cm, they were cut and transferred to rooting medium (containing 0.2 mg / L NAA, 10 mg / L Kan, and 400 mg / L Car) for screening;

[0038] (5) After the transformed seedlings have taken root and grown leaves, DNA is extracted from the transformed seedlings and identified by PCR;

[0039] (6) PCR identification was performed using the upstream primers of the 35S promoter of the plant expression vector and the downstream primers of the BnSME1 gene. The positive seedlings identified by PCR were transplanted into nutrient pots in an artificial climate greenhouse for growth. After the seeds matured, the individual plants were harvested to obtain T1 generation transgenic rapeseed seeds (identification results are as follows: Figure 2 shown).

[0040] 2. Use hygromycin to screen homozygous positive transgenic rapeseed plants. The specific steps are as follows:

[0041] (1) Disinfect T1 transgenic rapeseed seeds by referring to the experimental operation steps for the above-mentioned sterile rapeseed seedlings;

[0042] (2) Spread the sterilized seeds on MS solid medium containing 25 mg / L hygromycin, culture them in the dark in an incubator for about 5 days, and then switch to culture under normal light;

[0043] (3) When two true leaves grow, transplant the positive seedlings into nutrient pots in an artificial climate greenhouse;

[0044] (4) After the positive transgenic rapeseed seeds mature, harvest the seeds individually and store them in sequence to obtain T2 generation transgenic rapeseed seeds;

[0045] (5) The T2 generation transgenic rapeseed seeds were screened for hygromycin resistance according to the above experimental steps, and strains with a 3:1 ratio were screened and isolated. The strains were transplanted into an artificial climate greenhouse, and individual plants were harvested and numbered and stored in sequence to obtain T3 generation transgenic rapeseed seeds;

[0046] (6) The T3 generation transgenic rapeseed seeds were screened for hygromycin resistance according to the above experimental steps, and all normally growing strains were screened out to obtain homozygous T3 generation transgenic rapeseed.

[0047] Example 4 Analysis of Oil Content in BnSME1 Transgenic Rapeseed

[0048] Three homozygous T3 generation rapeseed BnSME1 transgenic lines were selected and the seed oil content was measured using a nuclear magnetic resonance instrument. The results showed that after increasing the expression of the BnSME1 gene in rapeseed through genetic engineering technology, the oil content of rapeseed seeds increased significantly, which can achieve an increase in rapeseed oil production. The experiment verified that compared with the control plants (untransformed plants), after overexpressing the BnSME1 gene, the oil content of the seeds of the three rapeseed BnSME1 transgenic lines increased by 11%, 6.13% and 16.26% respectively; the average oil content of rapeseed seeds increased by about 11.13% (such as Figure 3 shown).

Claims

1. Genes BnSME1 or its encoding protein or containing gene BnSME1 The use of an expression vector in increasing the oil content of rapeseed seeds is characterized in that, The coding sequence of the gene is shown in SEQ ID NO.

1.

2. Genes BnSME1 or its encoding protein or containing gene BnSME1 The application of the expression vector in the breeding of high oil-yield rapeseed is characterized in that, The coding sequence of the gene is shown in SEQ ID NO.

1.

3. A method for increasing the oil content of rapeseed seeds, characterized in that: Overexpression in rapeseed BnSME1 The coding sequence of the gene is shown in SEQ ID NO.1.

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