Application of GmHMGS gene and recombinant overexpression vector in regulating plant fatty acid content

Abstract

The present invention belongs to the technical field of genetic engineering, and particularly relates to an GmHMGS application of a gene and a recombinant overexpression vector in regulating the fatty acid content of plants, and the GmHMGS nucleotide sequence of the gene is shown as SEQ ID NO.1. The present invention uses an overexpression vector of an exogenous gene sequence to introduce the GmHMGS gene shown as SEQ ID NO.1 into plants for overexpression GmHMGS of the gene, and the plants show that the contents of palmitoleic acid, arachidonic acid, docosanoic acid and erucic acid are significantly higher than those of the wild type; at the same time, the contents of stearic acid, oleic acid and linolenic acid are significantly reduced. The present invention regulates the GmHMGS expression of the gene in plants and then uses it to cultivate new plant varieties with adjustable fatty acid content, which provides a new idea for cultivating new crop varieties and has great application value in molecular breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an GmHMGS application of a gene and a recombinant overexpression vector in regulating the fatty acid content of plants. Background Art

[0002] Soybean ( Glycine max (Linn.) Merr) is an annual herbaceous plant of the legume family. Its plant height is about 30 cm - 90 cm. It is the second largest source of vegetable oil raw materials in the world. Its seeds are rich in oil and protein, and have wide application values in the fields of medicine, food, etc. It plays an important role in reducing the incidence of cardiovascular diseases and the content of low-density cholesterol in the blood. In addition, as an important crop for both food and oil, it also plays a key role in the production of livestock and poultry products and the yield of soybean oil.

[0003] The synthesis of plant fatty acids is affected by various factors. For example, high temperature will increase the saturation degree of cell membranes, resulting in a decrease in the content of unsaturated fatty acids, and at the same time, it will also affect photosynthesis, thus inhibiting fatty acid synthesis; salt stress will damage the stability of cell membranes through ionic toxicity and osmotic stress, leading to the oxidation of unsaturated fatty acids in membrane lipids, and ultimately affecting fatty acid synthesis; in addition, pathogen infection will also change the fatty acid composition of plant cell membranes. In short, the synthesis of plant fatty acids is affected by various factors such as environment, nutrition, and biology. These factors ultimately inhibit fatty acid synthesis by interfering with the structure of plant cell membranes, affecting photosynthesis, and changing hormone balance. Based on this, the present invention proposes a new strategy for regulating the fatty acid content of plants. Summary of the Invention

[0004] To avoid the influence of environmental, nutritional, and biological factors on the fatty acid content of plants, the present invention provides an GmHMGS application of a gene in regulating the fatty acid content of plants.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The present invention provides an GmHMGS application of a gene in regulating the fatty acid content of plants, and the GmHMGS nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Preferably, the fatty acids include at least one of palmitoleic acid, adrenic acid, behenic acid, erucic acid, oleic acid, stearic acid, and linolenic acid; overexpressing the GmHMGS gene up-regulates palmitoleic acid, adrenic acid, behenic acid, and erucic acid; and down-regulates oleic acid, stearic acid, and linolenic acid.

[0008] The present invention provides a recombinant overexpression vector containing the GmHMGSRecombinant overexpression vector of gene.

[0009] The preparation method of the recombinant overexpression vector comprises the following steps:

[0010] Extract soybean RNA, reverse transcribe it into cDNA, and amplify GmHMGS gene using cDNA as a template;

[0011] Digest the overexpression vector with enzymes;

[0012] Connect GmHMGS gene with the digested overexpression vector to obtain the recombinant overexpression vector.

[0013] Preferably, the overexpression vector is pCAMBIA3301-eGFP.

[0014] Preferably, when digesting the overexpression vector, the restriction endonuclease used is Bam H I.

[0015] The present invention also provides an application of the recombinant overexpression vector, and the recombinant overexpression vector regulates the fatty acid content of plants by constructing transgenic plants overexpressing GmHMGS gene.

[0016] Preferably, the construction method of the transgenic plants overexpressing GmHMGS gene is as follows:

[0017] Transform the recombinant overexpression vector into Agrobacterium competent cells;

[0018] Perform enlarged culture to prepare an infection solution;

[0019] Use the infection solution to infect wild-type plants, and culture to obtain transgenic plants overexpressing GmHMGS gene.

[0020] Preferably, the Agrobacterium competent cells are GV3101.

[0021] Preferably, the OD 600 value of the infection solution is 0.8.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention provides an GmHMGS application of gene in regulating the fatty acid content of plants, and the nucleotide sequence of the GmHMGS gene is as shown in SEQ ID NO.1. The present invention uses an overexpression vector of an exogenous gene sequence to introduce the GmHMGS gene as shown in SEQ ID NO.1 into plants for overexpression GmHMGSGenes, and the plants showed significantly higher contents of palmitoleic acid, arachidonic acid, behenic acid and erucic acid than the wild type; at the same time, the contents of stearic acid, oleic acid and linolenic acid were significantly decreased. The present invention is used to cultivate new plant varieties with controllable fatty acid content by regulating the GmHMGS gene expression in plants, which provides a new idea for cultivating new crop varieties and has great application value in molecular breeding.

[0024] The present invention uses GmHMGS gene to construct a recombinant overexpression vector, and through Arabidopsis thaliana genetic transformation, explores the GmHMGS gene's influence on the metabolic pathways related to fatty acid synthesis, providing a new idea for plant molecular breeding improvement and increasing fatty acid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For GmHMGS gene amplification product electrophoresis pattern, M: DL2000bp DNA marker; -: negative control; Lanes 1-6 are GmHMGS gene's 6 parallel samples.

[0026] Figure 2 For pCAMBIA3301-eGFP single digestion product electrophoresis pattern, M: DL10000bp DNA marker; Lanes 1-4 are the single digestion results of 4 parallel samples of pCAMBIA3301-eGFP.

[0027] Figure 3 For GmHMGS gene's influence on key enzyme genes related to different fatty acid syntheses, A-J are LPAT2 , LACS8 , DGAT , ACC1 , FAD2 , LEC1 , LACS6 , FAD3 , LACS1 and FATB , and in the figure, * represents statistical difference of p<0.05, *** represents statistical difference of p<0.001, **** represents statistical difference of p<0.0001.

[0028] Figure 4 For fatty acid detection map of T2 generation Arabidopsis thaliana seeds, A and B are the detection results of two wild type Arabidopsis thaliana parallel plants; C and D are the detection results of two transgenic Arabidopsis thaliana parallel plants overexpressing GmHMGS gene. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described below through specific embodiments, but the scope of the present invention is not limited. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0030] The inventive concept of the present invention is as follows:

[0031] The synthesis of plant fatty acids is affected by various factors such as environment, nutrition, and organisms. These factors ultimately inhibit the synthesis of fatty acids by interfering with the plant cell membrane structure, affecting photosynthesis, and changing hormone balance. To avoid the influence of environmental, nutritional, and biological factors on plant fatty acid content, the present invention provides an GmHMGS application of a gene in regulating plant fatty acid content, and the GmHMGS nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0032] GmHMGS , that is, hydroxymethylglutaryl-CoA synthase gene, English name 3-Hydroxy-3- methylglutaryl-CoA synthetase gene , abbreviated as HMGS , is an important regulatory point in the metabolism of terpenoids. It has been reported that HMGS the gene can affect the contents of carbohydrates, amino acids, and fatty acids. It may indirectly affect the levels of soluble sugars and starch by regulating the primary metabolic pathway and participate in the extensive regulation of metabolic pathways. Therefore, based on transcriptome information, cloning and analyzing key regulatory genes of important agronomic traits are crucial for cultivating new high-yield and high-quality soybean varieties.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art. Among them, Escherichia coli DH5α competent cells were purchased from TransGen Biotech Co., Ltd.; Agrobacterium Agrobacterium tumefaciens GV3101 competent cells were purchased from TOLOBIO Biotechnology Co., Ltd.; the pCAMBIA3301-eGFP vector used for expression vector construction was stored in our laboratory. All kinds of drug reagents were purchased from Kangwei Reagent Biotechnology Co., Ltd. unless otherwise specified.

[0034] Example 1

[0035] GmHMGS The application of the gene and the recombinant overexpression vector in regulating plant fatty acid content is as follows:

[0036] 1. Construct a recombinant overexpression vector overexpressing the GmHMGS gene.

[0037] In this example, young pods 30 days after flowering of the soybean variety Jinong 18 "JN18" were used as materials to extract RNA, and the RNA was reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed with primer F with a homologous arm and primer R with a homologous arm to obtain a 1470 bp CDS sequence of the GmHMGS gene with a homologous arm. The PCR reaction system is shown in Table 1. GmHMGS The original sequence of the gene is shown in SEQ ID NO.1; the sequence of the amplified gene with a homologous arm GmHMGS is shown in SEQ ID NO.2. The sequence of primer F is shown in SEQ ID NO.3; the sequence of primer R is shown in SEQ ID NO.4.

[0038] SEQ ID NO.3: acacgctgagtgtcaggatccATGGCAAAGAATGTGGGGATC.

[0039] SEQ ID NO.4: tccaaacgcatgcagggatccTCAGTGGCCATTAGCAATGCC.

[0040] In the sequences of SEQ ID NO.3 and SEQ ID NO.4, the lowercase letters are the homologous arm sequences of seamless cloning, and the uppercase letters are the sequences of the 3´ end and 5´ end of the GmHMGS gene.

[0041] SEQ ID NO.1:

[0042]

[0043] SEQ ID NO.2:

[0044]

[0045] The lowercase letters in SEQ ID NO.2 are the homologous arm sequences for seamless cloning.

[0046] Table 1 PCR reaction system

[0047]

[0048] The size of the PCR product was identified by agarose gel electrophoresis and then recovered from the gel. The identification results are as Figure 1 shown.

[0049] Digest the overexpression vector pCAMBIA3301-eGFP with Bam H I. The results are as Figure 2 shown, and then recover from the gel; using the special recombinase 2×Basic Assembly Mix, ligate the above PCR product and the digested pCAMBIA3301-eGFP by homologous recombination. The detailed reaction system is shown in Table 2. React at 50 °C for 15 min in a PCR instrument. After the reaction, cool the ligation product on ice and then transfer it into Escherichia coli by heat shock method. Screen positive clones on LB medium containing kanamycin. Extract plasmids from the positive clones. After correct sequencing, the recombinant overexpression vector, pCAMBIA3301-eGFP- GmHMGS is successfully obtained.

[0050] Table 2 Homologous recombination reaction system

[0051]

[0052] 2. Preparation of transgenic Arabidopsis thaliana plants overexpressing GmHMGS genes.

[0053] 2.1. Use the recombinant overexpression vector pCAMBIA3301-eGFP- GmHMGS to transform Agrobacterium tumefaciens.

[0054] Take 1 μg of the recombinant overexpression vector pCAMBIA3301-eGFP- GmHMGS and add it to 100 μL of Agrobacterium tumefaciens competent cells GV3101. Gently tap the bottom of the tube to mix evenly. Incubate on ice for 10 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and in an ice bath for 5 min. Subsequently, add 900 μL of antibiotic-free YEP liquid medium and culture at 28 °C and 200 rpm for 2 h; spread evenly on LB medium containing 50 μg / mL kanamycin and culture in an inverted position at 28 °C for 2 days.

[0055] 2.2. Identification of Agrobacterium tumefaciens bacterial solution.

[0056] Pick a single colony and place it in 5 mL of LB medium containing 50 μg / mL rifampicin + 50 μg / mL kanamycin. Incubate overnight at 28°C with shaking at 200 rpm. Then, perform PCR verification on the Agrobacterium liquid using primer F-1 and primer R-1. The detailed PCR reaction system is shown in Table 3. The primer sequences of primer F-1 and primer R-1 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0057] SEQ ID NO.5: ATGGCAAAGAATGTGGGGATC.

[0058] SEQ ID NO.6: TCAGTGGCCATTAGCAATGCC.

[0059] Table 3 PCR reaction system for Agrobacterium identification

[0060]

[0061] 2.3 Preparation of the infection solution.

[0062] Collect the cells of the above Agrobacterium liquid and resuspend them with the resuspension solution to make the OD 600 value reach 0.8 to obtain the infection solution.

[0063] The formula of the resuspension solution is as follows:

[0064] Each liter of the resuspension solution contains 4.4 g of MS basal medium powder, 100 g of sucrose, 2 mL of 1000×B5 vitamins, 20 μL of 1 mg / mL 6-BA, 400 μL of Silweet L-77, 180 μL of 1 M NaOH, and 1950 mL of ddH2O.

[0065] After preparing the infection solution, invert the Arabidopsis thaliana at the flowering stage so that the entire inflorescence is completely immersed in the infection solution for 5 minutes. Then, carefully take out the Arabidopsis thaliana and place it sideways in a clean plastic tray, and cover it with a plastic film to avoid light and maintain humidity, and continue to culture for 24 h; subsequently, transfer it to normal light for cultivation. When the siliques of Arabidopsis thaliana are completely withered and about to crack after about 4 weeks, the seeds of Arabidopsis thaliana overexpressing GmHMGS the gene can be harvested. Plant the seeds of T0 generation Arabidopsis thaliana overexpressing GmHMGS the gene and continue with subsequent screening and identification.

[0066] Overexpression GmHMGS Screening and identification of transgenic Arabidopsis thaliana plants overexpressing the gene:

[0067] Spray Arabidopsis thaliana seedlings at the four-leaf stage with Basta solution once every two days. After some Arabidopsis thaliana seedlings turn yellow and wither, transplant the Arabidopsis thaliana in normal growth state to a new small pot to obtain Arabidopsis thaliana overexpressingGmHMGS For the transgenic Arabidopsis thaliana plants with the

[0068] gene, the rosette leaf genome was extracted later for PCR identification. The Basta used in this invention was purchased from Yuanye Bio-Technology Co., Ltd., and the product number was S18166. When preparing the Basta solution, the volume ratio of Basta to H2O was 1.5:1000.

[0069] 3. Overexpression GmHMGS The gene affects the expression levels of the key enzyme genes related to fatty acid synthesis.

[0070] The list of abbreviations in this invention is shown in Table 4.

[0071] Table 4 List of Abbreviations

[0072]

[0073] In this invention, the key enzyme genes related to fatty acid synthesis were detected by qRT-PCR FATB , FAD2 , LPAT2 , LACS8 , LACS1 , LACS6 , DGAT , ACC1 , LEC1 and FAD3 in transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana. Taking the changes in the expression levels of the key enzyme genes related to fatty acid synthesis in wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana overexpressing the GmHMGS gene as an index, cDNA from wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana overexpressing the GmHMGS gene was used as a template for qRT-PCR analysis, and specific primers were designed using NCBI. The expression levels of the key enzyme genes were detected by qRT-PCR in transgenic Arabidopsis thaliana overexpressing the GmHMGS gene and wild-type Arabidopsis thaliana in the T2 generation. The results are shown in Figure 3 . Compared with wild-type Arabidopsis thaliana, the expression levels of GmHMGS , AtLACS1 , AtLACS6 , AtLACS8 , AtFATB and AtFAD2 genes were significantly higher in transgenic Arabidopsis thaliana overexpressing the

[0074] The above results indicate that GmHMGS may directly or indirectly affect the expression levels of the key enzyme genes related to fatty acids, and then confirm that GmHMGS may be involved in the fatty acid synthesis metabolism of Arabidopsis thaliana.

[0075] 4. Overexpression GmHMGS The gene affects the fatty acid content.

[0076] The present invention detects the overexpression of T2 generation by the third method of GB 5009.168-2016 National Food Safety Standard for Determination of Fatty Acids in Food. GmHMGS The fatty acid content of transgenic Arabidopsis seeds and wild-type Arabidopsis seeds. Figure 4 , overexpression GmHMGS The palmitoleic acid, arachiddienoic acid, docosanoic acid and erucic acid in transgenic Arabidopsis thaliana were significantly higher than those in wild-type Arabidopsis thaliana, while the content of oleic acid, stearic acid and linolenic acid was significantly reduced. GmHMGS The relative percentage contents of palmitoleic acid, arachidienoic acid, behenic acid, erucic acid, oleic acid, stearic acid and linolenic acid in the transgenic Arabidopsis thaliana were 0.212%, 1.78%, 1.49%, 1.44%, 13.3%, 3.22% and 21.2%, respectively. Compared with the wild-type Arabidopsis thaliana, the contents of palmitoleic acid, arachidienoic acid, behenic acid and erucic acid increased by 30.06%, 15.58%, 154.70% and 10.77%, respectively, while the contents of oleic acid, stearic acid and linolenic acid decreased by 6.34%, 5.29% and 0.93%, respectively.

[0077] In summary, GmHMGS Genes involved in the regulation of plant fatty acid content.

[0078] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. GmHMGS Use of a gene in regulating the fatty acid content of plants, characterized in that, The said GmHMGS The nucleotide sequence of the gene is shown in SEQ ID NO.1; The application refers to regulating the fatty acid content of plants by constructing a recombinant overexpression vector and using the recombinant overexpression vector to construct transgenic plants overexpressing GmHMGS genes, and the recombinant overexpression vector contains the GmHMGS genes; The plant is *Arabidopsis thaliana*.

2. The application according to claim 1, characterized in that, The fatty acid includes at least one of palmitoleic acid, adrenic acid, behenic acid, erucic acid, oleic acid, stearic acid, and linolenic acid; Overexpressing the said GmHMGS gene upregulates palmitoleic acid, eicosadienoic acid, behenic acid, and erucic acid; and downregulates oleic acid, stearic acid, and linolenic acid.

3. The application according to claim 1, characterized in that, The method for preparing the recombinant overexpression vector comprises the following steps: Extract soybean RNA, reverse transcribe it into cDNA, and amplify the GmHMGS gene using the cDNA as a template; Digest the overexpression vector with restriction enzymes; Connect the GmHMGS gene with the digested overexpression vector to obtain a recombinant overexpression vector.

4. The application according to claim 3, characterized in that The overexpression vector is pCAMBIA3301-eGFP.

5. The application according to claim 3, characterized in that When digesting the overexpression vector, the restriction endonuclease used is Bam H I.

6. The application according to claim 1, wherein The overexpression GmHMGS The method for constructing a transgenic plant with the gene is as follows: Transform the recombinant overexpression vector into Agrobacterium competent cells; Perform enlarged culture to prepare an infection solution; Infect wild-type plants with an infection solution, culture them, and obtain transgenic plants overexpressing GmHMGS the gene.

7. The application according to claim 6, characterized in that, The Agrobacterium competent cells are GV3101.

8. The application according to claim 6, characterized in that, The OD of the infection solution 600 value is 0.8.

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