A gene related to coix seed oil synthesis, ClDGAT2, and its application.

Abstract

This invention relates to ClDGAT2, a gene related to oil synthesis in Job's tears, and its applications, belonging to the field of molecular biology. On one hand, this invention provides ClDGAT2, a gene related to oil synthesis in Job's tears, and its point-to-point mutant gene ClDGAT2T; on the other hand, it provides the application of the above genes in regulating plant oil accumulation. This invention is the first to transiently express ClDGAT2 and ClDGAT2T in tobacco leaves, finding that ClDGAT2T resulted in greater total oil production in tobacco leaves than ClDGAT2, indicating that in plants, mutating alanine (A) at amino acid position 204 of ClDGAT2 to serine (S) can enhance its oil synthesis capacity.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a gene ClDGAT2 related to the synthesis of coix seed oil and its application. Background Technology

[0002] Diacylglycerol acyltransferase (DGAT) is an enzyme that catalyzes the acylation of diacylglycerol (DAG) at the sn-3 position using fatty acyl-CoA molecules. There are four main types of this enzyme: diacylglycerol acyltransferase 1 (DGAT1), diacylglycerol acyltransferase 2 (DGAT2), diacylglycerol acyltransferase 3 (DGAT3), and wax ester synthase / diacylglycerol acyltransferase (WS / DGAT). Other similar enzymes have also been found in various plants. DGAT is a key rate-limiting enzyme for lipid accumulation in plants and has been extensively studied in transgenic plants to increase lipid yield. As the final enzyme in TAG biosynthesis, DGATs are crucial for oilseed development and have been targeted by genetic engineering to increase the yield of stored lipids in plants.

[0003] Job's tears (Coix seed) falls under the category of food and medicine with the same origin. Triglycerides are the main component of Job's tears oil, accounting for 86.96%–94.39% of the oil content. The triglycerides in Job's tears are mainly unsaturated fatty acid triglycerides; 1,3-dilinoleoyl-2-oleic acid (LOL) and 1-palmitoyl-2-oleoyl-3-glycerotrilinoleate (PLO) are the main active ingredients in Job's tears oil. Numerous studies have shown that Job's tears oil has various pharmacological effects, including improving liver function, anti-cancer properties, regulating intestinal flora, improving spleen function, and promoting urine excretion. Therefore, increasing the oil content of Job's tears is valuable.

[0004] Currently, conventional hybridization methods are commonly used to breed new biological varieties, but this method is too time-consuming. The rapidly developing genetic engineering technology offers new avenues for biological genetic improvement, and genetic transformation using genes that play a crucial role in lipid synthesis is an important means of obtaining high-oil-content germplasm. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for the coix seed oil synthesis-related gene ClDGAT2 and its application.

[0006] The present invention is implemented using the following technical solutions:

[0007] The first aspect of this invention provides a coix seed oil synthesis-related gene ClDGAT2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A second aspect of the present invention provides a protein encoded by the above-mentioned gene, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0009] The third aspect of the present invention provides a point-to-point mutant gene ClDGAT2T of the above-mentioned gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] The fourth aspect of the present invention provides a protein encoded by the above-mentioned mutant gene, the amino acid sequence of which is shown in SEQ ID NO.4.

[0011] The fifth aspect of this invention provides the application of the above-mentioned gene or the above-mentioned mutant gene in regulating plant oil accumulation.

[0012] The sixth aspect of the present invention provides a recombinant vector, expression cassette, and transgenic recombinant bacteria containing the above-mentioned gene or the above-mentioned mutated gene.

[0013] The seventh aspect of the present invention provides a method for regulating plant oil accumulation, wherein the above-mentioned mutant gene is constructed into a plant expression vector, which is introduced into plant cells to express the gene and enhance the accumulation of oil in the plant.

[0014] The present invention has the following beneficial effects:

[0015] 1. This invention is the first to obtain the ClDGAT2 gene and its encoded protein from Coix lacryma-jobi, and verifies its biological function in regulating lipid synthesis.

[0016] 2. This invention is the first to perform a single point mutation on the Coix lacryma-jobi ClDGAT2 gene and its encoded protein, and verifies its biological function in regulating lipid synthesis.

[0017] 3. This invention is the first to express and culture ClDGAT2 and ClDGAT2T in H1246 yeast. It was found that ClDGAT2T caused H1246 yeast to produce more lipids than ClDGAT2 caused H1246 yeast to produce more lipids. This indicates that in eukaryotic cells, mutating alanine (A) at amino acid position 204 of ClDGAT2 to serine (S) can enhance its lipid synthesis ability.

[0018] 4. This invention is the first to transiently express ClDGAT2 and ClDGAT2T in tobacco leaves. It was found that ClDGAT2T caused tobacco leaves to produce more total oil than ClDGAT2 caused tobacco leaves to produce more total oil. This indicates that in plants, mutating alanine (A) at amino acid position 204 of ClDGAT2 to serine (S) can enhance its ability to synthesize oil. Attached Figure Description

[0019] Figure 1Detection of positive H1246 yeast strains containing ClDGAT2 and ClDGAT2T;

[0020] Figure 2 Oil content of positive H1246 yeast strains containing ClDGAT2 and ClDGAT2T;

[0021] Figure 3 Nile red staining results of positive H1246 yeast strains containing ClDGAT2 and ClDGAT2T;

[0022] Figure 4 Nile red staining results and fatty acid content after transient overexpression of ClDGAT2 and ClDGAT2T in tobacco leaves. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific examples. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0024] Example 1: Cloning of the coding sequence (CDS) of diacylglycerol acyltransferase 2 (DGAT2) and construction of the plasmid

[0025] Total RNA was extracted from Coix seed using the RNeasy plant micro-extraction kit, and cDNA was obtained using an RNA reverse transcription kit. The coding sequence of ClDGAT2 was amplified using terminal-specific primers containing restriction enzyme sites and 2×Phanta FlashMaster MixP520 high-fidelity enzyme. The amplification system and procedure are shown in Tables 1 and 2. The seamless cloning primer sequence for ClDGAT2 is shown in Table 3. The nucleotide sequence of ClDGAT2, a gene related to Coix lipid synthesis, is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2.

[0026] Table 1 PCR buffer system

[0027]

[0028] Table 2 Amplification Procedure

[0029]

[0030] Primers used (Table 3)

[0031]

[0032] After digesting the pYES2.0 vector, the ClDGAT2 fragment was ligated to the vector digestion product using a seamless cloning kit, transformed into DH5α competent cells, positive clones were screened and sequenced, and plasmids were extracted after correct sequencing.

[0033] Example 2: Mutation of diacylglycerol acyltransferase 2 (DGAT2) and construction of plasmid

[0034] A single amino acid residue point-to-point mutation was introduced into Coix seed ClDGAT2 using the Mut Express II rapid site-directed mutagenesis kit V2, resulting in a mutation of alanine (A) to serine (S) at amino acid position 204 of ClDGAT2, thus constructing the point-to-point mutant gene ClDGAT2T. The nucleotide sequence of the point-to-point mutant gene ClDGAT2T is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.4. Primers for the single point mutation are shown in Table 3.

[0035] The mutant gene ClDGAT2T was ligated into the pYES2.0 vector and transformed into DH5α competent cells. Positive clones were screened and sequenced. Plasmids were extracted after successful sequencing. Primers for positive colony PCR screening are shown in Table 3.

[0036] Example 3: Construction and culture of yeast expression vector

[0037] The ClDGAT2-pYES2.0 plasmid constructed in Example 1, the ClDGAT2T-pYES2.0 plasmid constructed in Example 2, and the empty vector control were transformed into Saccharomyces cerevisiae strain H1246 using the lithium acetate-mediated transformation method. The transformed strains were cultured on solid SC-Ura medium supplemented with 2% glucose, and positive H1246 yeast were screened by PCR.

[0038] Positive H1246 yeast cells were inoculated into liquid SC-Ura medium containing 2% galactose and 1% raffinose, respectively, and induced in the dark for 5 days under shaking conditions at 30°C and 180 rpm until the stationary phase was reached. After centrifuging at 4000 rpm for 5 minutes to remove the culture medium, the collected yeast cells were freeze-dried. Finally, the completely dried yeast cells were ground into powder for subsequent experiments.

[0039] Example 4: Lipid Analysis

[0040] Weigh 50 mg of each freeze-dried yeast material into a mortar, add a small amount of quartz sand, grind thoroughly into a fine powder, transfer to a 50 mL centrifuge tube, add 7.5 mL of methanol:chloroform (V:V = 2:1) solution, and extract in a constant temperature shaker at 37°C for 24 h. After a short centrifugation, collect the upper organic phase. Add another 7.5 mL of methanol:chloroform (V:V = 2:1) solution to the remaining sample precipitate, continue extraction for 12 h, and collect the upper organic phase after centrifugation. Repeat the above step once. Combine the three organic phases, add 5 mL of chloroform solution and 9 mL of 1% sodium chloride solution to make the final chloroform:methanol:water volume ratio 2:2:1.8. Mix thoroughly and centrifuge at 8000 rpm for 8-10 min. Recover the lower organic phase and transfer it to a pre-weighed finger-shaped glass tube. Purge with nitrogen and weigh again (total weight of the finger-shaped glass tube and yeast fatty acids). Total fatty acid content of yeast = (total weight after extraction - weight of tube before extraction) / 0.05.

[0041] Fatty acid analysis of tobacco leaves: Weigh 30 mg of tobacco leaves, add 1.5 mL of 2.5% H2SO4, 0.4 mL of toluene, and 150 mL of 2 mg / mL C7:0 standard. Incubate in a 90°C water bath for 1 hour, then cool the sample to room temperature, mix with 1.8 mL of distilled water and 1 mL of n-hexane, and centrifuge at 2000 rpm / min for 5 minutes. Transfer the upper n-hexane layer to a GC-MS sample vial and allow to stand for analysis.

[0042] In the gas chromatograph, hydrogen was used as the carrier gas at a flow rate of 1.0 mL / min. The inlet temperature, transfer tube temperature, and ion source temperature were maintained at 220℃, 230℃, and 230℃, respectively. The column oven temperature was increased from 60℃ to 180℃ at a rate of 10℃ / min, and then from 180℃ to 21℃ at a rate of 3℃ / min.

[0043] Example 5: Nile Red Staining

[0044] A suitable amount of cells in the stationary phase (400 μL) was collected by centrifugation, washed twice with 1×PBS, gently mixed in 20 μL of PBS, and then mixed with 5 μL of Nile Red at a concentration of 1 μg / μL. The stained cells were incubated at 30°C in the dark for 10 minutes, then washed twice with 1×PBS and diluted in 100 μL of 1×PBS. The fluorescence of Nile Red-stained lipids was observed at an excitation wavelength of 488 nm and an emission wavelength between 560 and 620 nm. The stained cells were observed and photographed using a fluorescence microscope equipped with a digital camera; fluorescence indicated the formation of lipids.

[0045] Example 6: Transient expression of ClDGAT2 and ClDGAT2T in tobacco leaves

[0046] Enzyme restriction sites were added to the 5' ends of the upstream and downstream primers of the two original genes (ClDGAT2) to construct overexpression plasmid primers (Table 3). The recombinant plasmids constructed in Examples 1 and 2 were used as templates for PCR. The products were ligated into the pCAMBIA1301 vector to obtain overexpression plasmids of the original and mutagenic genes.

[0047] After sequencing, the correct recombinant plasmid was transformed into Agrobacterium GV3101(pSoup). Positive clones of Agrobacterium were inoculated into 50 mL of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin) and cultured overnight at 220 rpm in a shaker at 28°C. The next morning, 0.75 μL of acetylsylgenin (100 mM) was added and the culture was continued for 2 hours. The organisms were then collected by centrifugation at 4,000 rpm for 10 minutes, and the supernatant was discarded. The organisms were resuspended in wetting buffer (5 mM MgSO4, 100 mM acetylsylgenin, 5 mM MES) to achieve an OD600 of 0.6. Single positive Agrobacterium clones were then injected into the underside of tobacco leaves (4-6 leaf stage) using a needle-free syringe. After overnight culture in the dark, the tobacco was transferred to a light incubator the next day and cultured for 4-5 days before Nile Red staining.

[0048] Results analysis:

[0049] like Figure 1 As shown in the figure, the H1246 yeast strain containing ClDGAT2 showed a band, indicating that the ClDGAT2-pYES2 plasmid was successfully transformed into H1246 yeast.

[0050] like Figure 2 As shown, the lipid content of positive H1246 yeast strains ClDGAT2 and ClDGAT2T showed a highly significant difference, indicating that mutating alanine (A) at amino acid position 204 of ClDGAT2 to serine (S) can significantly enhance its lipid synthesis ability.

[0051] like Figure 3 As shown, the Nile Red staining results of the positive H1246 yeast strains containing ClDGAT2 and ClDGAT2T indicate that the introduction of both ClDGAT2 and ClDGAT2T can successfully induce H1246 yeast to produce lipids.

[0052] like Figure 4As shown, Nile red staining results in tobacco leaves overexpressing ClDGAT2 and ClDGAT2T showed that the number of oil droplets in leaves overexpressing ClDGAT2T was significantly higher than that in leaves overexpressing ClDGAT2. Simultaneously, the content of various fatty acids in leaves overexpressing ClDGAT2T was significantly higher than that in leaves overexpressing ClDGAT2. This indicates that mutating alanine (A) to serine (S) at amino acid position 204 of ClDGAT2 can significantly enhance its lipid synthesis capacity. The nucleotide sequence of ClDGAT2 is shown in SEQ ID NO.1.

[0053] The amino acid sequence of the protein encoded by ClDGAT2 is shown in SEQ ID NO.2:

[0054]

[0055]

[0056] The nucleotide sequence of ClDGAT2T is shown in SEQ ID NO.3:

[0057] The amino acid sequence of the ClDGAT2T encoded protein is shown in SEQ ID NO.4:

[0058]

[0059]

Claims

1. A gene related to the synthesis of coix oil ClDGAT2 characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The protein encoded by the gene of claim 1, wherein, The amino acid sequence of the encoded protein is shown as SEQ ID NO.

2.

3. A point-to-point mutated gene of claim 1. ClDGAT2T characterized in that The nucleotide sequence of the mutant gene is shown as SEQ ID NO.

3.

4. The protein encoded by the mutant gene of claim 3, wherein, The amino acid sequence of the encoded protein is shown as SEQ ID NO.

4.

5. The gene of claim 1 or the mutant gene of claim 3 is used for regulating the accumulation of oil in tobacco leaves.

6. A recombinant vector, an expression cassette, a transgenic recombinant bacteria containing the gene of claim 1 or the mutant gene of claim 3.

7. A method of modulating lipid accumulation in a plant, comprising, The mutant gene of claim 3 is constructed into a plant expression vector, introduced into tobacco leaf cells, expressed in tobacco leaves and promotes the accumulation of oil in tobacco leaves.

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