gma-miR287, recombinant overexpression vector and its application in regulating plant fatty acid content
By constructing a recombinant overexpression vector of gma-miR287, regulating plant fatty acid synthesis, solving the problem of inhibiting fatty acid synthesis by environmental factors, achieving controllable regulation of fatty acid content, and providing a new pathway for improved molecular breeding.
Patent Information
- Application Number
- CN202510502571.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2045-04-22
AI Technical Summary
Plant fatty acid synthesis is affected by environmental, nutritional and biological factors, resulting in synthesis inhibition, and the existing technology lacks effective regulatory means.
Gma-miR287 microRNA was used to regulate plant fatty acid synthesis, and the content of plant fatty acids was regulated by constructing a recombinant overexpression vector.
It significantly regulates the content of plant fatty acids, increases the content of palmitoleic acid, linolenic acid, eicodacidic acid, benzyl acid and erucic acid, and reduces the content of palmitic acid, oleic acid, linoleic acid and eicodacidic acid, providing new ideas for molecular breeding improvement.
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Figure CN120026028B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a gma-miR287, recombinant overexpression vector and its application 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, rich in protein and oil, and is the second largest source of plant oil raw materials in the world. The oil rich in its seeds is not only an important substance for maintaining life activities, a component of cells, and the main form of energy storage, but also 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, and at the same time have wide application values in the fields of medicine, food, etc.
[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 will also affect photosynthesis, thereby 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, changing hormone balance, etc. 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 a gma-miR287.
[0005] The technical solution adopted by the present invention is as follows:
[0006] In the first aspect of the present invention, there is provided a gma-miR287 , and the gma- miR287 has a nucleotide sequence as shown in SEQ ID NO.1.
[0007] In the second aspect of the present invention, there is provided a recombinant overexpression vector containing the gma-miR287 .
[0008] In the third aspect of the present invention, there is provided a preparation method of the recombinant overexpression vector, including the following steps:
[0009] Extract soybean RNA, reverse transcribe it into cDNA, and amplify using cDNA as a templategma-miR287 ; Digest the overexpression vector with restriction enzymes; gma-miR287 Connect with the digested overexpression vector to obtain a recombinant overexpression vector.
[0010] Preferably, the overexpression vector is pCAMBIA3301-eGFP.
[0011] Preferably, when digesting the overexpression vector, the restriction endonuclease used is Bam H I.
[0012] The fourth aspect of the present invention provides an application of the gma-miR287 or the recombinant overexpression vector, and the application refers to regulating the content of plant fatty acids by constructing an overexpressing gma-miR287 plant.
[0013] Preferably, the fatty acids include at least one of palmitic acid, oleic acid, linoleic acid, eicosenoic acid, palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; overexpressing the gma-miR287, up-regulates palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; down-regulates palmitic acid, oleic acid, linoleic acid and eicosenoic acid.
[0014] Preferably, the method for constructing the overexpressing gma-miR287 plant is as follows:
[0015] Transform the recombinant overexpression vector into Agrobacterium competent cells; expand the culture to prepare an infection solution; use the infection solution to infect wild-type plants and culture to obtain an overexpressing gma-miR287 plant.
[0016] Preferably, the Agrobacterium competent cells are GV3101.
[0017] Preferably, the OD 600 value of the infection solution is 0.8.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] The present invention provides a gma-miR287 for regulating the content of plant fatty acids, and the nucleotide sequence of the gma-miR287 is shown in SEQ ID NO.1. The present invention uses the young pods of the 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 genes. By constructing a recombinant overexpression vector and performing Arabidopsis thaliana genetic transformation, explore gma-miR287The influence of genes on the fatty acid synthesis and metabolism-related pathways expands the reports on miRNAs related to soybean fatty acid synthesis, providing new ideas for plant molecular breeding improvement and increasing fatty acid content. The present invention imports the gma-miR287 gene sequence shown in SEQ ID NO.1 into plants, overexpresses the gma-miR287 gene, and the plants show significantly reduced contents of palmitic acid, oleic acid, linoleic acid, and eicosenoic acid, and significantly increased contents of palmitoleic acid, linolenic acid, eicosenoic acid, behenic acid, and erucic acid. By regulating the gma-miR287 gene sequence expression in plants, the present invention is used to cultivate new plant varieties with controllable fatty acid content, providing new ideas for cultivating new crop varieties and having great application value in molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 is gma-miR287 the electrophoresis map of the amplification product, M: DL2000bp DNA marker; lanes 1 to 6 are gma- miR287 6 parallel samples.
[0021] Figure 2 is the electrophoresis map of the single digestion product of pCAMBIA3301-eGFP, M: DL10000bp DNA marker; lanes 1 to 4 are the single digestion results of 4 parallel samples of pCAMBIA3301-eGFP.
[0022] Figure 3 is gma-miR287 the influence on key enzyme genes related to different fatty acid syntheses, A to J are LACS8 , LPAT2 , DGAT , LACS1, ACC1 , LEC1 , FATB, FAD2 , LACS6 and FAD3 , * indicates p a statistical difference of <0.05, ** indicates p a statistical difference of <0.01, *** indicates p a statistical difference of <0.0001, **** indicates p a statistical difference of <0.0001.
[0023] Figure 4 is the fatty acid detection map of the seeds of Arabidopsis thaliana overexpressing gma-miR287 in the T3 generation, A and B are the detection results of two wild-type Arabidopsis thaliana parallel plants; C and D are the detection results of two Arabidopsis thaliana parallel plants overexpressing gma-miR287 . DETAILED DESCRIPTION OF THE INVENTION
[0024] 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.
[0025] The inventive concept of the present invention is as follows:
[0026] 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 a method for regulating plant fatty acid content gma- miR287, The gma-miR287 has a nucleotide sequence as shown in SEQ ID NO.1.
[0027] gma-miR287 is a microRNA. MicroRNA, abbreviated as miRNA, is a non-coding RNA with a length of about 18nt - 25nt, which is highly conserved and tissue-specific and plays a key role in plant gene expression regulation. miRNA has a core regulatory function in 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. Currently, the research on miRNA in oil synthesis and its genetic connection with oil characteristics is relatively scarce, and most research focuses on key enzyme genes.
[0028] 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 with reference to 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, the competent cells of Escherichia coli DH5α were purchased from TransGen Biotech; the competent cells of Agrobacterium Agrobacterium tumefacien s GV3101 were purchased from TOLOBIO Biotech; the pCAMBIA3301-eGFP vector for overexpression vector construction was stored in our laboratory. All kinds of drug reagents were purchased from Kangwei Reagent Biotechnology Co., Ltd. unless otherwise specified.
[0029] Example 1
[0030] gma-miR287 A recombinant overexpression vector and its application in regulating plant fatty acid content are as follows:
[0031] 1. A method for regulating the fatty acid content in plants gma-miR287.
[0032] In this invention, the young pods of the soybean low linolenic acid mutant "MT72" and its wild type "JN18" at 30 days and 40 days after flowering were used as materials for transcriptome sequencing analysis, and differentially expressed fatty acid-related genes were screened and successfully cloned gma-miR287 , and its nucleotide sequence is shown as SEQ ID NO.1.
[0033] SEQ ID NO.1:
[0034] ccaugauugaugcauAACUGAAAUUCUUAAAGCAuuccuaauuuucacaucauuuugaugaugggaaugcuuuaagaauuuccaguuagguaucaagac.
[0035] The sequence of SEQ ID NO.1 is gma-miR287 the precursor sequence of gma-miR287 and the capital letters represent gma-miR287 the mature sequence of
[0036] 2. A recombinant overexpression vector for regulating the fatty acid content in plants.
[0037] In this example, the young pods of the soybean variety Jinong 18 "JN18" at 30 days after flowering were used as materials, RNA was extracted, and the RNA was reverse transcribed into cDNA. Using the cDNA as a template, PCR amplification was performed with primer F with homologous arms and primer R with homologous arms to obtain gma-miR287 the sequence with homologous arms. The PCR reaction system is 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.
[0038] Primer F, SEQ ID NO.3:
[0039] acacgctgagtgtcaggatccCCATGATTGATGCATAACTGAAA.
[0040] Primer R, SEQ ID NO.4:
[0041] tccaaacgcatgcagggatccGTCTTGATACCTAACTGGAAA.
[0042] The lowercase letters in SEQ ID NO.3 and SEQ ID NO.4 are seamless cloning homologous arm sequences, and the capital letters are gma-miR287 Sequences at the 3' and 5' ends.
[0043] Through the above process, the cloned gene sequence containing homologous arms gma-miR287 has a length of 141 bp, and the sequence information is shown in SEQ ID NO.2.
[0044] SEQ ID NO.2:
[0045] acacgctgagtgtcaggatccccatgattgatgcatAACTGAAATTCTTAAAGCAttcctaattttcacatcattttgatgatgggaatgctttaagaatttccagttaggtatcaagacggatccctgcatgcgtttgga, where the capital letters represent gma-miR287 the mature sequence of gma-miR287 and the lowercase part represents the stem-loop structure of
[0046] Table 1 PCR reaction system
[0047]
[0048] The size of the PCR product was identified by agarose gel electrophoresis and then gel-extracted. The identification results are as Figure 1 shown.
[0049] The overexpression vector pCAMBIA3301-eGFP was digested with Bam H I single enzyme, and the results are as Figure 2 shown, and then gel-extracted; using a special recombinase 2×Basic Assembly Mix, the above PCR product and the digested pCAMBIA3301-eGFP were ligated by homologous recombination. The detailed reaction system is shown in Table 2. React at 50 °C in a PCR instrument for 15 min. After the reaction, cool the ligation product on ice; transfer it to Escherichia coli by heat shock and screen for positive clones on LB medium containing kanamycin. Plasmid extraction was performed, and after sequencing, the recombinant overexpression vector pCAMBIA3301-eGFP- gma- miR287 .
[0050] Table 2 Homologous recombination reaction system
[0051]
[0052] 3. Application of a recombinant overexpression vector for regulating plant fatty acid content.
[0053] 3.1 Preparation of plants overexpressing gma-miR287 .
[0054] The present invention obtained overexpressed Arabidopsis thaliana plants by using a recombinant overexpression vector, including the following steps: gma-miR287 as follows:
[0055] 3.1.1. Use the recombinant overexpression vector pCAMBIA3301-eGFP- gma-miR287 to transform Agrobacterium tumefaciens.
[0056] Take 1 μg of the recombinant overexpression vector pCAMBIA3301-eGFP- gma-miR287 and add it to 100 μL of Agrobacterium tumefaciens competent cells GV3101. Gently tap the bottom of the tube to mix evenly. Then, let it stand on ice for 10 min, in liquid nitrogen for 5 min, in a water bath at 37 °C for 5 min, and in an ice bath for 5 min. After that, add 900 μL of antibiotic-free YEP liquid medium and culture at 28 °C and 200 rpm for 2 h. Spread it evenly on an LB medium containing 50 μg / mL kanamycin and culture it upside down at 28 °C for 2 days.
[0057] 3.1.2. Identification of Agrobacterium tumefaciens liquid.
[0058] Pick a single colony and inoculate it into 5 mL of LB medium containing 50 μg / mL rifampicin + 50 μg / mL kanamycin. Culture it at 28 °C and 200 rpm overnight, and perform PCR verification on the Agrobacterium tumefaciens 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.
[0059] Primer F-1, SEQ ID NO.5: ccatgattgatgcataactgaaa.
[0060] Primer R-1, SEQ ID NO.6: gtcttgatacctaactggaaa.
[0061] Table 3 PCR reaction system for identifying Agrobacterium tumefaciens liquid
[0062]
[0063] 3.1.3. Preparation of the infection solution.
[0064] Collect the cells of the above Agrobacterium tumefaciens liquid and resuspend them with a resuspension solution to make its OD 600 value reach 0.8 to obtain the infection solution.
[0065] The formula of the resuspension solution is:
[0066] Each resuspension 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.
[0067] 3.1.4. Preparation of overexpressing gma-miR287 plants.
[0068] 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, cover it with a plastic film to avoid light and maintain humidity, and culture it for 24 hours; 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 overexpressing gma-miR287 Arabidopsis thaliana seeds can be harvested. Plant the T0 generation of overexpressing gma-miR287 Arabidopsis thaliana seeds and continue with subsequent screening and identification.
[0069] Screening and identification of overexpressing gma-miR287 Arabidopsis thaliana plants:
[0070] Spray the four-leaf stage seedlings with Basta solution once every two days. After some Arabidopsis thaliana seedlings turn yellow and withered, transplant the normally growing Arabidopsis thaliana into new small pots to obtain overexpressing gma-miR287 Arabidopsis thaliana plants, and later extract the rosette leaf genome for PCR identification.
[0071] The Basta used in this invention is purchased from Yuanye Bio-Technology Co., Ltd., and the product number is S18166; when preparing the Basta solution, the volume ratio of Basta to H2O is 1.5:1000.
[0072] 3.2. Overexpressing gma-miR287 affects the expression levels of key enzyme genes related to fatty acid synthesis.
[0073] The abbreviation list of this invention is shown in Table 4.
[0074] Table 4 Abbreviation list
[0075]
[0076] This invention detects the key enzyme genes related to fatty acid synthesis through qRT-PCR FATB , FAD2 , LPAT2 , LACS8 , LACS1 , LACS6 , DGAT , ACC1 ,LEC1 and FAD3 in overexpressing gma-miR287 Arabidopsis plants and wild-type Arabidopsis thaliana. Using the expression level changes of wild-type Arabidopsis thaliana and overexpressing gma-miR287 Arabidopsis plants as indicators, qRT-PCR analysis was performed using the cDNA of wild-type Arabidopsis thaliana and overexpressing gma-miR287 Arabidopsis plants as templates, and specific primers were designed using NCBI. The expression levels of key enzyme genes in T2 generation overexpressing gma-miR287 plants and wild-type plants were detected by qRT-PCR. The results are shown in Figure 3 In overexpressing gma-miR287 Arabidopsis plants, LACS8 , DGAT , ACC1, LEC1, FATB, FAD2, LACS6 and FAD3 showed significantly lower expression levels than those in wild-type plants. This indicates that gma- miR287 may directly or indirectly affect the expression levels of key enzyme genes related to fatty acids, thus confirming that gma-miR287 may be involved in the fatty acid synthesis and metabolism of Arabidopsis thaliana.
[0077] 3.3. Overexpression of gma-miR287 affects fatty acid content.
[0078] In this invention, the fatty acid contents in the seeds of T3 generation overexpressing gma-miR287 plants and the seeds of wild-type Arabidopsis thaliana plants were detected by the third method of GB 5009.168-2016 National Food Safety Standard Determination of Fatty Acids in Foods.
[0079] The results are shown in Figure 4 In overexpressing gma-miR287 plants, the contents of palmitic acid, oleic acid, linoleic acid, and eicosenoic acid were significantly lower than those in wild-type Arabidopsis thaliana; at the same time, the contents of palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid, and erucic acid were significantly increased. 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 overexpressing gma-miR287 plants were 7.49%, 13.5%, 29.2%, 18.9%, 0.176%, 22.7%, 1.95%, 1.23%, and 1.44% in turn. Compared with wild-type Arabidopsis thaliana, palmitic acid, oleic acid, linoleic acid, and eicosenoic acid were decreased by 3.23%, 4.93%, 1.68%, and 5.5% respectively; palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid, and erucic acid were increased by 7.96%, 6.07%, 26.62%, 110.26%, and 10.77% respectively.
[0080] The results show that gma-miR287May be involved in the regulation of plant fatty acid content.
[0081] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.
[0082] The above-described embodiments only express several implementation manners of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention.
Claims
1. gma-miR287 An application for regulating the fatty acid content in plants, characterized in that The said gma-miR287 has a nucleotide sequence as shown in SEQ ID NO.1, and the said gma-miR287 is a microRNA; The application refers to regulating the content of plant fatty acids by constructing plants overexpressing the gma-miR287 ; The fatty acids include palmitic acid, oleic acid, linoleic acid, eicosenoic acid, palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; Overexpressing the said gma-miR287, Up-regulating palmitoleic acid, linolenic acid, eicosadienoic acid, behenic acid and erucic acid; down-regulating palmitic acid, oleic acid, linoleic acid and eicosenoic acid; The plant is Arabidopsis thaliana.
2. A recombinant overexpression vector comprising the one described in claim 1 gma-miR287 .
3. The recombinant overexpression vector according to claim 2, wherein The method for constructing a plant overexpressing the following gma-miR287 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 plants overexpressing the gma-miR287 .
4. The recombinant overexpression vector according to claim 3, wherein The Agrobacterium competent cells are GV3101.
5. The recombinant overexpression vector according to claim 3, characterized in that, The OD of the infection solution 600 is 0.
8.
6. The preparation method of the recombinant overexpression vector according to claim 2, characterized in that, It includes the following steps: Extract soybean RNA, reverse transcribe it into cDNA, and amplify using the cDNA as a template gma-miR287 ; Digest the overexpression vector with restriction enzymes; Ligate gma-miR287 with the digested overexpression vector to obtain a recombinant overexpression vector.
7. The preparation method according to claim 6, characterized in that, The overexpression vector is pCAMBIA3301-eGFP.
8. The preparation method according to claim 6, characterized in that, When digesting the overexpression vector, the restriction endonuclease used is Bam H I.