Gma-miR287, recombinant overexpression vector and application of recombinant overexpression vector in regulating and controlling content of vegetable fatty acid

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gma-miR287, a recombinant overexpression vector and application of the gma-miR287 to regulation and control of the content of vegetable fatty acid, and the nucleotide sequence of the gma-miR287 is shown as SEQ ID NO.1. The invention further discloses a preparation method of the gma-miR287. According to the invention, the fatty acid related gene gma-miR287 with differential expression is screened and successfully cloned. According to the invention, the gma-miR287 is introduced into a plant to obtain a plant with overexpressed gma-miR287, and the plant shows that the contents of palmitic acid, oleic acid, linoleic acid and eicosaenoic acid are reduced, and the contents of palmitoleic acid, linolenic acid, eicosaenoic acid, behenic acid and erucic acid are increased. The expression of the gma-miR287 in the plant is regulated and controlled so as to be used for cultivating a new plant variety with the adjustable and controlled fatty acid content, a thought is provided for cultivating the new plant variety, and the gma-miR287 has great application value in molecular breeding.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to a gma-miR287, Recombinant overexpression vector and its application in regulating plant fatty acid content. Background Art

[0002] Soybean ( Glycine max Linn. Merr) is an annual leguminous herbaceous plant, rich in protein and oil, and is the world's second largest source of vegetable oil raw materials. The oil rich in its seeds is not only an important substance for maintaining life activities, a component of cells and a major form of energy storage, but its metabolite fatty acids play an important role in reducing the incidence of cardiovascular diseases and the content of low-density cholesterol in the blood. It also has a wide range of application value in the fields of medicine and food.

[0003] The synthesis of plant fatty acids is affected by many factors, for example: high temperature will increase the saturation of cell membranes, resulting in a decrease in the content of unsaturated fatty acids, and will also affect photosynthesis, thereby inhibiting fatty acid synthesis; salt stress will destroy the stability of cell membranes through ion poisoning and osmotic stress, leading to the oxidation of unsaturated fatty acids in membrane lipids, and ultimately affecting the synthesis of fatty acids; in addition, pathogen infection will also change the fatty acid composition of plant cell membranes. In short, plant fatty acid synthesis is affected by many factors such as the environment, nutrition, and biology, which ultimately inhibit the synthesis of fatty acids 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 content of plant fatty acids. Summary of the invention

[0004] In order to prevent environmental, nutritional and biological factors from affecting the content of plant fatty acids, the present invention provides a method for regulating the content of plant fatty acids. gma-miR287.

[0005] The technical solution adopted by the present invention is: The first aspect of the present invention provides a method for regulating the content of plant fatty acids. gma-miR287 , gma- miR287 The nucleotide sequence is shown in SEQ ID NO.1.

[0006] A second aspect of the present invention provides a method comprising gma-miR287 recombinant overexpression vector.

[0007] The third aspect of the present invention provides a method for preparing the recombinant overexpression vector, comprising the following steps: Extract soybean RNA, reverse transcribe into cDNA, and amplify using cDNA as template gma-miR287 ; digest the overexpression vector;gma-miR287 The recombinant overexpression vector was obtained by connecting it with the overexpression vector after enzyme digestion.

[0008] Preferably, the overexpression vector is pCAMBIA3301-eGFP.

[0009] Preferably, when the overexpression vector is cleaved, the restriction endonuclease used is Bam HI.

[0010] A fourth aspect of the present invention provides a gma-miR287 Or the application of the recombinant overexpression vector, the application refers to constructing an overexpression vector gma-miR287 Regulates the content of plant fatty acids in plants.

[0011] Preferably, the fatty acid comprises at least one of palmitic acid, oleic acid, linoleic acid, eicosenoic acid, palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; overexpression of the gma-miR287, Palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid were upregulated; while palmitic acid, oleic acid, linoleic acid and eicosenoic acid were downregulated.

[0012] Preferably, the overexpression gma-miR287 The method for constructing the plant is as follows: The recombinant overexpression vector is transformed into Agrobacterium competent cells; the culture is expanded to prepare an infection solution; the infection solution is used to infect wild-type plants, culture, and obtain an overexpression vector. gma-miR287 of plants.

[0013] Preferably, the Agrobacterium competent cell is GV3101.

[0014] Preferably, the OD of the infection solution is 600 The value is 0.8.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for regulating the content of plant fatty acids gma-miR287 , gma-miR287 The nucleotide sequence of is shown in SEQ ID NO.1. The present invention uses young pods of soybean low linolenic acid mutant "MT72" and its wild type "JN18" 30 days and 40 days after flowering as materials for transcriptome sequencing analysis, screens and successfully clones differentially expressed fatty acid-related gma-miR287 By constructing a recombinant overexpression vector and performing genetic transformation of Arabidopsis thaliana, we explored gma-miR287 The influence of genes on fatty acid synthesis and metabolism pathways has expanded the report of miRNA related to soybean fatty acid synthesis, providing a new idea for plant molecular breeding and improvement and increasing fatty acid content.gma-miR287 Gene sequences are introduced into plants and overexpressed gma-miR287 The plant showed that the content of palmitic acid, oleic acid, linoleic acid and eicosapentaenoic acid was significantly reduced, and the content of palmitoleic acid, linolenic acid, eicosapentaenoic acid, behenic acid and erucic acid was significantly increased. gma-miR287 The expression of gene sequences in plants is then used to breed new plant varieties with regulated fatty acid content, which provides new ideas for breeding new crop varieties and has great application value in molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for gma-miR287 Electrophoresis of amplified products, M: DL2000bp DNA marker; Lane 1 to Lane 6 are gma- miR287 6 parallel samples.

[0017] Figure 2 The electrophoresis diagram of the single restriction enzyme digestion product of pCAMBIA3301-eGFP, M: DL10000bp DNA marker; lanes 1 to 4 are the single restriction enzyme digestion results of 4 parallel samples of pCAMBIA3301-eGFP.

[0018] Figure 3 for gma-miR287 Effects on key enzyme genes related to the synthesis of different fatty acids, A~J are LACS8 , LPAT2 , DGAT , LACS1, ACC1 , LEC1 , FATB, FAD2 , LACS6 and FAD3 ,*express p Statistical difference <0.05, ** indicates p *** indicates statistical difference <0.01 p Statistical difference <0.0001, **** indicates p Statistical difference <0.0001.

[0019] Figure 4 T 3 Overexpression gma-miR287 Fatty acid detection profiles in Arabidopsis seeds. A and B are the detection results of two parallel strains of wild-type Arabidopsis; C and D are the detection results of two overexpressed gma-miR287 Results of testing of Arabidopsis thaliana parallel strains. DETAILED DESCRIPTION

[0020] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0021] The inventive concept of the present invention is as follows: Plant fatty acid synthesis is affected by multiple factors such as environment, nutrition and biology. These factors interfere with plant cell membrane structure, affect photosynthesis, change hormone balance, etc., and ultimately inhibit fatty acid synthesis. In order to avoid environmental, nutritional and biological factors affecting plant fatty acid content, the present invention provides a method for regulating plant fatty acid content. gma- miR287, Said gma-miR287 The nucleotide sequence is shown in SEQ ID NO.1.

[0022] gma-miR287 It is a kind of microRNA, abbreviated as miRNA, which is a non-coding RNA with a length of about 18nt~25nt. It is highly conserved and tissue-specific, and plays a key role in the regulation of plant gene expression. miRNA has core regulatory functions for plant growth, development, hormone synthesis and signal transduction, and stress response. When facing environmental stress, plants enhance stress resistance by regulating the expression of specific miRNAs. In addition, miRNA may also affect the synthesis and accumulation of plant oils by regulating the expression of genes related to lipid metabolism. At present, research on miRNA in oil synthesis and its genetic connection with oil characteristics is relatively scarce, and most studies focus on key enzyme genes.

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention and implement it, the present invention is 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 Quanshijin Biotechnology Co., Ltd.; Agrobacterium Agrobacterium tumefacien s GV3101 competent cells were purchased from TOLOBIO Biotechnology Co., Ltd.; the pCAMBIA3301-eGFP vector used for overexpression vector construction was preserved by our laboratory, and various drugs and reagents were purchased from Kangwei Reagent Biotechnology Co., Ltd. unless otherwise specified.

[0024] Example 1 gma-miR287 , recombinant overexpression vector and its application in regulating plant fatty acid content, as follows: 1. A method for regulating the content of plant fatty acids gma-miR287.

[0025] The present invention uses the young pods of soybean low linolenic acid mutant "MT72" and its wild type "JN18" 30 days and 40 days after flowering as materials for transcriptome sequencing analysis, screening and successfully cloning differentially expressed fatty acid-related genes gma-miR287 , whose nucleotide sequence is shown in SEQ ID NO.1.

[0026] SEQ ID NO.1: ccaugauugaugcauAACUGAAAUUCUUAAAGCAuuccuaauuuucacaucauuuugaugaugggaaugcuuuaagaauuuccaguuagguaucaagac.

[0027] The sequence of SEQ ID NO.1 is gma-miR287 The precursor sequence of gma-miR287 The lowercase part represents the mature sequence of gma-miR287 stem-loop structure.

[0028] 2. A recombinant overexpression vector for regulating the content of plant fatty acids.

[0029] In this example, young pods of soybean variety Jinong 18 "JN18" 30 days after flowering were used as materials, RNA was extracted, and RNA was reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed using primers F with homology arms and primers R with homology arms to obtain cDNA with homology arms. gma-miR287 The sequences and PCR reaction system are shown in Table 1. The primer sequences of primer F and primer R are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0030] Primer F, SEQ ID NO.3: acacgctgagtgtcaggatccCCATGATTGATGCATAACTGAAA.

[0031] Primer R, SEQ ID NO.4: tccaaacgcatgcagggatccGTCTTGATACCTAACTGGAAA.

[0032] The lowercase letters in SEQ ID NO.3 and SEQ ID NO.4 are seamless clone homology arm sequences, and the uppercase letters are gma- miR287 The sequences at the 3´ and 5´ ends of the DNA sequence.

[0033] After the above process, the cloned gma-miR287The gene sequence length is 141 bp, and the sequence information is shown in SEQ ID NO.2.

[0034] SEQ ID NO.2: acacgctgagtgtcaggatccccatgattgatgcatAACTGAAATTCTTAAAGCAttcctaattttcacatcattttgatgatgggaatgctttaagaatttccagttaggtatcaagacggatccctgcatgcgtttgga, where capital letters represent gma-miR287 The lowercase part represents the mature sequence of gma-miR287 stem-loop structure.

[0035] Table 1 PCR reaction system

[0036] The PCR product was identified by agarose gel electrophoresis and then recovered from the gel. Figure 1 shown.

[0037] The overexpression vector pCAMBIA3301-eGFP was used Bam HI single enzyme digestion, the result is as follows Figure 2 As shown, the gel was recovered; the PCR product and the pCAMBIA3301-eGFP after enzyme digestion were connected by homologous recombination using a special recombinase 2×Basic Assembly Mix. The detailed reaction system is shown in Table 2. The reaction was carried out at 50°C for 15 minutes in a PCR instrument. After the reaction, the connection product was placed on ice for cooling; the product was transferred to Escherichia coli by heat shock method, and positive clones were screened on LB medium containing kanamycin. Plasmid extraction was performed, and the recombinant overexpression vector pCAMBIA3301-eGFP- gma- miR287 .

[0038] Table 2 Homologous recombination reaction system

[0039] 3. Application of a recombinant overexpression vector for regulating plant fatty acid content.

[0040] 3.1. Preparation of overexpression gma-miR287 of plants.

[0041] The present invention utilizes the recombinant overexpression vector to obtain the overexpression gma-miR287 thaliana plants, comprising the following steps: 3.1.1. Using the recombinant overexpression vector pCAMBIA3301-eGFP-gma-miR287 Transformation of Agrobacterium.

[0042] Take 1 μg of recombinant overexpression vector pCAMBIA3301-eGFP- gma-miR287 Add to 100 μL Agrobacterium competent cells GV3101, gently stir the bottom of the tube to mix, let stand on ice for 10 min, liquid nitrogen for 5 min, 37°C water bath for 5 min, add 900 μL antibiotic-free YEP liquid culture medium after ice bath for 5 min, and culture at 28°C, 200 rpm for 2 h; spread evenly on LB culture medium containing 50 μg / mL kanamycin, and culture upside down at 28°C for 2 days.

[0043] 3.1.2. Identification of Agrobacterium tumefaciens culture fluid.

[0044] A single colony was picked and placed in 5 mL LB medium containing 50 μg / mL rifampicin + 50 μg / mL kanamycin, cultured overnight at 28°C, 200 rpm, and the Agrobacterium bacterial solution was verified by PCR using primers F-1 and R-1. The detailed PCR reaction system is shown in Table 3. The primer sequences of primers F-1 and R-1 are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0045] Primer F-1, SEQ ID NO.5: ccatgattgatgcataactgaaa.

[0046] Primer R-1, SEQ ID NO.6: gtcttgatacctaactggaaa.

[0047] Table 3 PCR reaction system for Agrobacterium bacterial liquid identification

[0048] 3.1.3. Preparation of infection solution.

[0049] Collect the cells of the Agrobacterium bacterial solution and resuspend them in a resuspension solution to obtain an OD 600 When the value reaches 0.8, the infection solution is obtained.

[0050] The formula of the resuspension solution is: Each resuspension contained 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 ddH 2 O 1950mL.

[0051] 3.1.4. Preparation of overexpression gma-miR287 of plants.

[0052] After the infection solution is prepared, turn the Arabidopsis thaliana plants that are in the flowering stage upside down so that the entire inflorescence is completely immersed in the infection solution for 5 minutes. After that, carefully take out the Arabidopsis thaliana, place it on its side in a clean plastic tray, and cover it with plastic film to avoid light and maintain humidity. Continue to culture for 24 hours; then, transfer it to normal light for culture. After about 4 weeks, when the Arabidopsis thaliana siliques are completely withered and about to crack, the overexpression can be harvested. gma-miR287 Arabidopsis seeds. 0 Overexpression gma-miR287 The Arabidopsis seeds were planted for subsequent screening and identification.

[0053] Overexpression gma-miR287 Screening and identification of Arabidopsis plants: Use Basta solution to spray the seedlings at the four-leaf stage once every two days. After some Arabidopsis seedlings turn yellow, transplant the Arabidopsis seedlings in normal growth state into new small pots to obtain the overexpression gma-miR287 The Arabidopsis plants were cultured and the rosette leaf genome was extracted for PCR identification.

[0054] The Basta used in the present invention was purchased from Yuanye Biotechnology with the product number S18166. When preparing the Basta solution, Basta and H 2 The volume ratio of O is 1.5:1000.

[0055] 3.2 Overexpression gma-miR287 Affects the expression levels of key enzyme genes related to fatty acid synthesis.

[0056] The abbreviations of the present invention are shown in Table 4.

[0057] Table 4 Abbreviations

[0058] The present invention detects key enzyme genes related to fatty acid synthesis by qRT-PCR FATB , FAD2 , LPAT2 , LACS8 , LACS1 , LACS6 , DGAT , ACC1 , LEC1 and FAD3 In overexpression gma-miR287 Relative expression of Arabidopsis thaliana plants and wild-type Arabidopsis thaliana. gma-miR287 The expression level changes of Arabidopsis plants were used as indicators, and wild-type Arabidopsis and overexpressing gma-miR287 qRT-PCR analysis was performed using cDNA of Arabidopsis thaliana plants as templates, and specific primers were designed using NCBI. 2Overexpression gma-miR287 The expression levels of key enzyme genes in plants and wild-type plants. Figure 3 , in overexpression gma-miR287 In Arabidopsis plants, LACS8 , DGAT , ACC1, LEC1, FATB, FAD2, LACS6 and FAD3 The expression level of gma- miR287 It may directly or indirectly affect the expression of key fatty acid-related enzyme genes, which then confirms gma-miR287 May be involved in fatty acid synthesis in Arabidopsis thaliana.

[0059] 3.3 Overexpression gma-miR287 Affects fatty acid content.

[0060] The present invention detects T by the third method of GB 5009.168-2016 National Food Safety Standard for the Determination of Fatty Acids in Food. 3 Overexpression gma-miR287 Fatty acid content in seeds of plants and seeds of wild-type Arabidopsis plants.

[0061] Results Figure 4 , overexpression gma-miR287 The contents of palmitic acid, oleic acid, linoleic acid and eicosapentaenoic acid in the plants were significantly lower than those in wild-type Arabidopsis; meanwhile, the contents of palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid were significantly increased. gma-miR287 The relative percentage contents of palmitic acid, oleic acid, linoleic acid, eicosenoic acid, palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid in the seeds of the plants were 7.49%, 13.5%, 29.2%, 18.9%, 0.176%, 22.7%, 1.95%, 1.23% and 1.44%, respectively. Compared with the wild-type Arabidopsis, the contents of palmitic acid, oleic acid, linoleic acid and eicosenoic acid decreased by 3.23%, 4.93%, 1.68% and 5.5%, respectively; the contents of palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid increased by 7.96%, 6.07%, 26.62%, 110.26% and 10.77%, respectively.

[0062] The results show that gma-miR287 May be involved in the regulation of plant fatty acid content.

[0063] 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.

[0064] 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. A method for regulating the content of plant fatty acids gma-miR287 , characterized in that, Said gma-miR287 The nucleotide sequence is shown in SEQ ID NO.1, gma-miR287 It is a microRNA.

2. A method comprising the method according to claim 1 gma-miR287 recombinant overexpression vector.

3. The method for preparing a recombinant overexpression vector according to claim 2, characterized in that: The following steps are involved: Extract soybean RNA, reverse transcribe into cDNA, and amplify using cDNA as template gma-miR287 ; Enzyme digestion of overexpression vector; Will gma-miR287 The recombinant overexpression vector was obtained by connecting it with the overexpression vector after enzyme digestion.

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

5. The preparation method according to claim 3, characterized in that: When the overexpression vector is digested, the restriction endonuclease used is Bam HI.

6. As claimed in claim 1 gma-miR287 Or the use of the recombinant overexpression vector according to claim 2, characterized in that, The application refers to constructing an overexpression gma-miR287 Regulates the content of plant fatty acids in plants.

7. The use according to claim 6, characterized in that The fatty acid comprises at least one of palmitic acid, oleic acid, linoleic acid, eicosenoic acid, palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; Overexpression gma-miR287, Palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid were upregulated; while palmitic acid, oleic acid, linoleic acid and eicosenoic acid were downregulated.

8. The use according to claim 6, characterized in that Overexpression gma-miR287 The method for constructing the plant is as follows: Transforming the recombinant overexpression vector into Agrobacterium competent cells; Expand the culture and prepare the infection fluid; The wild-type plants are infected with the infection solution, cultured, and the overexpression of the gma-miR287 of plants.

9. The use according to claim 8, characterized in that The Agrobacterium competent cell is GV3101.

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

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