Oilseed rape seed oil content gene BnSME1 and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene for regulating and controlling the oil content of rape seeds and application. The gene for regulating and controlling the oil content of the oilseed rape seeds is BnSME1, after the gene is over-expressed in oilseed rape, the oil content of the seeds in a transgenic oilseed rape strain is increased compared with that of a contrast, and the maximum amplification of the oil content of the transgenic oilseed rape seeds is 16.26%. Therefore, the gene provided by the invention provides a new gene resource for high-oil breeding of the oilseed rape, and can be applied to improvement of the oil yield of the oilseed rape.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular 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 human diet structure, an important industrial raw material and bioenergy, and play an important role in national economic and social development. In recent years, with people's pursuit of healthy diet, plant oils and fats have gradually replaced animal oils and fats and become the main consumer oils in my country's edible oil market. Rapeseed is an important oil-bearing economic crop in my country, and rapeseed oil accounts for 40% of my country's edible oil; and rapeseed, as a winter crop, does not compete with rice and other grain crops for time and land. At the same time, rapeseed can be used for edible rapeseed, sightseeing, tourism, beekeeping, and green manure. It is an oil-bearing economic crop with great development potential. However, my country's rapeseed varieties have low oil content, low yield, and unstable quality. Therefore, increasing the oil yield of rapeseed is an important measure to ensure my country's edible oil supply. The increase in rapeseed oil production per unit area can be achieved by increasing yield and oil content. However, current studies have shown that oil content contributes more to rapeseed oil production. Every 1% increase in the oil content of rapeseed seeds is equivalent to a 2.5% increase in yield. Therefore, improving 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 the oil content of rapeseed seeds can reach up to 75%, but the average oil content of existing rapeseed varieties in my country is relatively low, and there is still much room for improvement. Rapeseed oil content is a quantitative trait controlled by multiple micro-effect genes and greatly affected by environmental conditions, which makes it very difficult to significantly increase the oil content of rapeseed seeds using traditional breeding techniques. At present, with the continuous development and maturity of molecular biology and sequencing technology, the mining, isolation, cloning and functional research of rapeseed seed oil content regulatory genes are becoming more and more in-depth; the use of genetic engineering technology and gene editing technology 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 as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.

[0006] The present invention also provides an amplification primer pair for the above gene, the amplification primer pair 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 basic vector of the plant expression vector includes 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 invention also provides application of the gene BnSME1 or its plant expression vector in improving the oil content of rapeseed seeds.

[0010] The present invention also provides a method for increasing the oil content of rapeseed seeds, which comprises overexpressing the BnSME1 gene in rapeseed, wherein 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: the present invention obtains the gene BnSME1 that can regulate the oil content of plant seeds through gene isolation and cloning, construction of expression vector and rapeseed transformation. The gene BnSME1 is applied to crop breeding. It is verified by the examples that after overexpression of the BnSME1 gene in rapeseed, the oil content of seeds in different transgenic rapeseed lines is increased compared with the control (non-transgenic plants), and the highest increase in the oil content of transgenic rapeseed seeds is 16.26%. It can be seen that the gene of the present invention provides a new gene resource for rapeseed high-oil breeding and can be used to increase the oil yield of rapeseed. 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 measuring the oil content of BnSME1 transgenic rapeseed seeds. DETAILED DESCRIPTION

[0015] The present invention provides a gene BnSME1 for regulating the oil content of rapeseed seeds, and the nucleotide sequence of the gene BnSME1 is shown in SEQ ID NO.1. In order to further illustrate the present invention, the gene BnSME1 for regulating the oil content of rapeseed seeds and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments. Although the above embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all embodiments. Those skilled in the art can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

[0016] In the following examples of the present invention, the experimental material used is 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 the 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 at both ends of the restriction site for PCR, and the PCR reaction system is based on 50 μL, preferably including: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, BnSME1-F 2 μL, BnSME1-R 2 μL, cDNA 1 μL of Zhongshuang No. 6 rapeseed and ddH 2 O 20μL. The PCR amplification procedure of the present invention preferably includes: 98°C pre-denaturation for 30s; 94°C denaturation for 10s, 65°C annealing for 5s, 72°C extension for 30s, 35 cycles; 72°C extension for 1min; and finally storage at 4°C. The amplified PCR product is purified using a PCR product purification kit and stored for later use. The plant overexpression vector pCAMBIA1301 is double-digested with BglII and PmlI restriction endonucleases, and after detection by 1% agarose gel electrophoresis, it is recovered and stored using a gel recovery kit.

[0019] The PCR purified product of the BnSME1 gene and the recovered restriction plasmid were recombined using a ligase-independent single-fragment rapid cloning kit. The reaction system was: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 50-200 ng of linearized vector, 10-200 ng of inserted fragment, and finally supplemented with ddH 2 0 to 20 μL. The recombinant product was transformed into E. coli DH5α competent cells, and after inverted culture in a 37°C incubator for 12 to 16 hours, the grown single clones were detected by PCR using the upstream primer of the vector 35S promoter (SEQ ID NO.5: 5'-acccacgaggagcatcgtgg-3') and the downstream primer of the terminator NOS (SEQ ID NO.6: 5'-gataatcatcgcaagaccgg-3'). The single clones that were positive after PCR verification were sent to Wuhan Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0020] The sequencing results show that the present invention has 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 pCAMBIA1301-BnSME1 recombinant plasmid and add it to 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 about 700 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), place in a shaker at 28 °C and shake at 200 rpm for 2 to 3 h;

[0026] (4) Spread the bacterial solution on LB solid medium supplemented with 50 mg / L kanamycin (Kan), 50 mg / L gentamicin (Gent), and 50 mg / L rifampicin (Rif), and culture in an inverted incubator at 28°C for 36 to 48 h;

[0027] (5) Perform PCR detection on the grown Agrobacterium single clones using the upstream primers of the 35S promoter of the plant expression vector and the downstream primers of the terminator NOS. Shake the positive single clones until OD 600 =1.8-2.0, and then preserve the bacteria in 50% glycerol and store in -80℃ ultra-low temperature refrigerator for later use.

[0028] 2. Preparation of Agrobacterium Suspension with 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 place in a 28°C shaker at 200 rpm until 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 It is about 1.0, and is prepared into 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 selected by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences) seeds were soaked in 70% ethanol solution for 1 min and then added with mercuric chloride (HgCl 2 ) solution for 13-15 minutes, washed with sterile water 5 times, spread on MS culture medium, and placed in an artificial climate culture room for growth to obtain rapeseed sterile seedlings for use;

[0035] (2) Take the cotyledon petioles of sterile seedlings of 4 to 5 days old, place them in the above-mentioned Agrobacterium suspension carrying the pCAMBIA1301-BnSME1 vector and soak them for 5 to 8 minutes, shake them gently, then pour out the bacterial solution, and absorb the bacterial solution remaining 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 to 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 they 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 (AgNO 3 ), 400 mg / L Car and 15 mg / L Kan) for screening, until the explants differentiate into regenerated green shoots;

[0037] (4) When the regenerated shoots grew to 1 cm, the shoots 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 of the transformed seedlings is extracted and PCR identification is performed;

[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 were as follows: Figure 2 shown).

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

[0041] (1) Referring to the experimental operation steps of the above-mentioned rapeseed sterile seedlings, the T1 generation transgenic rapeseed seeds were disinfected;

[0042] (2) Spread the sterilized seeds on MS solid medium containing 25 mg / L hygromycin, culture them in a dark 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, the seeds are harvested individually and numbered and stored in sequence to obtain T2 generation transgenic rapeseed seeds;

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

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

[0047] Example 4 Analysis of oil content in BnSME1 transgenic rapeseed

[0048] Three homozygous T3 generation rapeseed BnSME1 transgenic strains 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, and the oil yield of rapeseed can be increased. Experimental verification showed that compared with the control plants (untransformed plants), after overexpressing the BnSME1 gene, the oil content of seeds of the three rapeseed BnSME1 transgenic strains increased by 11%, 6.13% and 16.26% respectively; the oil content of rapeseed seeds increased by an average of about 11.13% (such as Figure 3 shown).

Claims

1. Genes that regulate oil content in rapeseed seeds BnSME1 , characterized in that, The coding sequence of the gene is shown in SEQ ID NO.

1.

2. The gene according to claim 1 BnSME1 The encoded protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the gene according to claim 1 BnSME1 expression vector.

4. The gene according to claim 1 BnSME1 Or use of the protein according to claim 2 or the expression vector according to claim 3 in increasing the oil content of rapeseed seeds.

5. The gene according to claim 1 BnSME1 Or use of the protein according to claim 2 or the expression vector according to claim 3 in breeding high-oil-yield rapeseed.

6. 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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