Application of gma-miR396b gene in regulating plant fatty acid synthesis

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to the application of gma-miR396b gene in regulating plant fatty acid synthesis. The nucleotide sequence of the gma-miR396b gene provided by the present invention is shown in SEQ ID NO.1. The present invention uses wild-type soybean "JN18" and low linolenic acid mutant "MT72" as materials, takes young pods 30d, 40d and 50d after soybean flowering for transcriptome sequencing, screens and successfully clones differentially expressed gma-miR396b precursor sequences related to soybean fatty acids, verifies the interaction between gma-miR396b and long-chain acyl-CoA synthetase through 3′PPM-RACE experiments, constructs an overexpression vector, and carries out genetic transformation of Arabidopsis to verify its function.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of gma-miR396b gene in regulating plant fatty acid synthesis. Background Art

[0002] Soybean, Glycine maxMerrAs an important oil crop in the world, soybean oil synthesis plays an important role in output value, and its content and quality are directly related to the market value of soybean. In particular, the composition of fatty acids in oil affects the nutritional value and industrial application of soybean oil. Therefore, studying the biosynthesis mechanism of soybean oil and fatty acids is of great significance to increasing the content of soybean oil and improving the quality of soybean oil.

[0003] MicroRNA, miRNA is a class of non-coding single-stranded RNA short sequences widely present in eukaryotes. Many recent new research results show that the miRNA396 family has relevant predictions and research on the adaptability of plant seeds and roots, affecting crop yield and stress resistance, but whether miRNA396b is involved in soybean fatty acid biosynthesis and how it affects soybean fatty acid synthesis has not been reported. Summary of the invention

[0004] In order to solve the problem in the prior art that whether miRNA396b is involved in soybean fatty acid biosynthesis and how it affects soybean fatty acid synthesis has not been reported, in order to achieve the above purpose, the present invention adopts the following technical solution:

[0005] The present invention uses young pods of soybean variety "JN18" and its low linolenic acid mutant "MT72" 30d, 40d and 50d after flowering as experimental materials, constructs 18 mRNA libraries and 18 miRNA libraries respectively, performs transcriptome and miRNA sequencing, and screens out the gma-miR396b gene related to soybean fatty acid biosynthesis.

[0006] The present invention provides the use of the gma-miR396b gene in regulating plant fatty acid synthesis, wherein the nucleotide sequence of the gma-miR396b gene is shown in SEQ ID NO.1. The precursor sequence of the gene is 169 bp, see Figure 1 , gma-miR396b specifically recognizes the target gene: long-chain acetyl-CoA synthetase 1 gene, abbreviated as ACLS1 gene.

[0007] Preferably, the primers for amplifying the gma-miR396b gene include gma-miR396b seamless-F and gma-miR396b seamless-R;

[0008] Among them, the nucleotide sequence of gma-miR396b seamless-F is as shown in SEQ ID NO.3: acgggggactcttgaccatggCTCAAGTCCTGGTCATGCTTTTC.

[0009] The nucleotide sequence of gma-miR396b seamless-R is shown in SEQ ID NO.4: ggggaaattcgagctggtcaccAGCCTGAATTGCCATATTCTCC.

[0010] Preferably, the gma-miR396b gene is used to construct an expression vector, and the expression vector is transferred into a plant to regulate the fatty acid content in the plant.

[0011] Preferably, the expression vector is obtained by inserting the gma-miR396b gene into the NcoI and BstEII restriction sites of the pCAMBI3301 vector.

[0012] Specifically, the method for constructing an expression vector containing the gma-miR396b gene comprises the following steps:

[0013] The correctly sequenced gma-miR396b plasmid was used as a template for amplification;

[0014] The obtained amplified product was double-digested with NcoI and BstEII double-digested pCAMBI3301 vector, and then transformed. The correct positive clone plasmid was extracted and verified, which was the expression vector pCAMBIA3301-miR396b.

[0015] The expression vector can be used to infect plant cells, study the function of foreign genes in plant tissue cells, and further study the function of miR396b gene in soybean fatty acid biosynthesis.

[0016] Preferably, the steps of amplifying using the pCAMBIA3301-miR396b plasmid as a template are:

[0017] Using pCAMBIA3301-miR396b plasmid as a template, gma-miR396b seamless-F and gma-miR396b seamless-R were used as primer pairs for amplification; wherein, the nucleotide sequence of gma-miR396b seamless-F is shown in SEQ ID NO.3; the nucleotide sequence of gma-miR396b seamless-R is shown in SEQ ID NO.4.

[0018] Preferably, the gma-miR396b gene increases the fatty acid content in plants by regulating the synthesis of fatty acids.

[0019] Preferably, the plants include soybean and Arabidopsis thaliana.

[0020] Preferably, overexpression of the gma-miR396b gene in plants can reduce the oleic acid content and increase the linolenic acid and eicosapentaenoic acid contents. 3 The relative percentage of oleic acid in Arabidopsis seeds overexpressing the gma-miR396b gene was 13.793%, while the relative percentage of oleic acid in wild-type seeds was 16.1%. 3 The relative percentage of linolenic acid in Arabidopsis seeds overexpressing the gma-miR396b gene was 20.9475%, while the relative percentage of linolenic acid in wild-type seeds was 19.8%. 3 The relative percentage of eicosaenoic acid in the seeds of Arabidopsis thaliana overexpressing the gma-miR396b gene was 20.1645%, while the relative percentage of eicosaenoic acid in the wild-type seeds was 18.6%.

[0021] Preferably, the gma-miR396b gene is overexpressed in Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana containing gma-miR396b.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention introduces the gma-miR396b gene with the coding sequence shown in SEQ ID NO.1 into the plant by using an exogenous gene expression vector, overexpresses the gene, and the plant shows an increase in oleic acid content, a decrease in linolenic acid content, and a decrease in eicosapentaenoic acid content. That is, the present invention finds through research that the miRNA396b gene can participate in the biosynthesis of soybean fatty acids, and by regulating the expression of the gma-miR396b gene in plants, it is used to cultivate new plant varieties with regulatable fatty acid content, providing a new idea for soybean molecular breeding improvement and increasing fatty acid content.

[0024] The present invention uses young pods of soybean low-linolenic acid mutant "MT72" and its wild type "JN18" at 30d, 40d, and 50d after flowering as materials for transcriptome sequencing analysis, screens and successfully clones differentially expressed fatty acid-related gma-miR396b precursor sequences, constructs overexpression vectors, and explores the effects of gma-miR396b on fatty acid anabolism-related pathways through Arabidopsis genetic transformation, expands the reporting of soybean fatty acid synthesis-related miRNAs, and provides new ideas for soybean molecular breeding improvement and increasing fatty acid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is the electrophoresis diagram of the gma-miR396b gene amplification product in the present invention, wherein M: DL2000 DNA marker; 1-3 are all target genes.

[0026] Figure 2 This is a schematic diagram of the interaction relationship between gma-miR396b gene cutting XM_006588497:ACLS1 in the present invention.

[0027] Figure 3 The electrophoresis diagram of the double enzyme digestion product of pCAMBIA3301-miR396b in the present invention, wherein M: DL10000 DNA marker; 1: single enzyme digestion result; 2: double enzyme digestion result.

[0028] Figure 4 It is the effect of the gma-miR396b gene on the expression of regulatory factors related to the key genes for fat regulation in the present invention, * indicates a statistical difference of p<0.05, and ** indicates a statistical difference of p<0.01; wherein, A is the relative expression of FAD2: fatty acid dehydrogenase; B is the relative expression of FATB: acyl-ACP thioesterase B; C is the relative expression of LACS8: long-chain acyl-CoA synthetase; D is the relative expression of LPAT2: lysophosphatidic acid acyltransferase 2.

[0029] Figure 5 T in the present invention 2 Fatty acid detection profiles in transgenic seeds of Arabidopsis thaliana, where A is the detection profile of wild-type Arabidopsis thaliana; B is the detection profile of Arabidopsis thaliana overexpressing the gma-miR396b gene sequence. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Escherichia coliDH5α competent cells were purchased from Quanshijin Biotechnology Co., Ltd.; AgrobacteriumEHA105 competent cells were purchased from Beijing Zhuangmeng International Biological Co., Ltd.; the pCAMBIA3301 vector used in the construction of the expression vector was produced by our laboratory; it was preserved in the Important Crop Gene Mining and Planting Innovation and Creation Laboratory of Jilin Agricultural University; various drugs and reagents were purchased from Kangwei Reagent Biotechnology Co., Ltd. unless otherwise specified.

[0032] The present invention provides an application of a gma-miR396b gene in plants, wherein the application is to regulate the synthesis of soybean fatty acids. The nucleotide sequence of the gma-miR396b gene precursor is shown in SEQ ID NO.1.

[0033] In some embodiments of the invention, the plant is Arabidopsis thaliana.

[0034] The present invention also provides a method for regulating the synthesis of soybean fatty acids, comprising regulating the expression of the gma-miR396b gene in a plant, wherein the nucleotide sequence of the gma-miR396b gene is shown in SEQ ID NO.1:

[0035] ctcaagtcctggtcatgcttTTCCACAGCTTTCTTGAACTTcttatgcatcttatatctctccacctccaggattttaagccctagaaGCTCAAGAAAGCTGTGGGAGAatatggcaattcaggct.

[0036] The cloned target gene: including homology arms, 169 bp as shown in SEQ ID NO.2:

[0037] ACGGGGGACTCTTGACCATGGctcaagtcctggtcatgcttTTCCACAGCTTTCTTGAACTTcttatgcatcttatatctctccacctccaggattttaagccctagaaGCTCAAGAAAGCTGTGGGAGAatatggcaattcaggctGGTGACCAGCTCGAATTTCCCC.

[0038] In some embodiments of the present invention, the following steps are included:

[0039] (1) The interaction relationship between the target gene and ACLS1, a long-chain acyl-CoA synthetase, was verified by 3′PPM-RACE experiment;

[0040] (2) constructing a recombinant expression vector, inserting the gma-miR396b gene of the gene sequence shown in SEQ ID NO.1 into a plant expression vector to form a recombinant expression vector;

[0041] (3) transforming the recombinant expression vector into cells of the target plant to overexpress the gma-miR396b gene sequence to obtain a candidate plant;

[0042] (4) Screening resistant plants from the candidate plants to obtain plants related to soybean fatty acids.

[0043] In some embodiments of the present invention, the overexpression vector is pCAMBIA3301.

[0044] In some embodiments of the present invention, the transformation is carried out using the inflorescence infection method.

[0045] In some embodiments of the invention, the plant is Arabidopsis thaliana.

[0046] Example 1

[0047] 1. Acquisition of gma-miR396b gene

[0048] Preliminary transcriptome sequencing based screening of gma-miR396b

[0049] The soybean variety "JN18" was used as a control, and the low linolenic acid mutant "MT72" obtained by EMS mutagenesis in the laboratory was used as a material. At the end of April 2021, they were planted in the experimental field of the Biotechnology Center of Jilin Agricultural University. Fresh young pods of "JN18" and "MT72" were collected 30d, 40d, and 50d after soybean flowering, and total RNA was extracted for sequencing. The differential miRNA expression pattern was analyzed, and the miRNAs related to fatty acid synthesis and their target genes were mined. Through GO and KEGG analysis, gma-miR396 with high differential expression and involved in soybean fatty acid biosynthesis was mined as a candidate gene. The enrichment of predicted genes in cellular components, molecular functions, and biological processes was completed through the Gene Ontology database: http: / / www.geneo ntolo gy.org. The functional analysis and prediction of differently expressed genes was performed through the Kyoto Encyclopedia of Genes and Genomes database: http: / / www.genome.jp / kegg / .

[0050] Among them, the soybean variety "JN18" is a high-oil and high-yield soybean variety bred by the Biotechnology Center of Jilin Agricultural University. It is the main soybean variety currently promoted in Jilin Province.

[0051] Mutant “MT72” Mutant MT72 is the fourth generation low linolenic acid mutant material obtained from JN18 in 2015 after being subjected to 0.5% EMS mutation for 6h.

[0052] The total RNA was extracted by using TRIzon Reagent TRIZOL method to extract total RNA from soybean young pods, and the steps were as shown in the instruction manual.

[0053] Soybean genome extraction

[0054] 100 mg of fresh soybean "MT72" leaves were collected as materials. The specific experimental steps are shown in the instructions of the NuClean Plant Genomic DNA Kit. The extracted genome was tested for sample quality and concentration using a gel electrophoresis instrument and an ultraviolet spectrophotometer.

[0055] PCR amplification of target gene

[0056] Using soybean genomic DNA as a template, the gma-miR396b gene cloning PCR system and PCR conditions are shown in Table 1 and Table 2:

[0057] Table 1 PCR reaction system

[0058]

[0059] Table 2 PCR reaction conditions

[0060]

[0061] Purification and recovery of target fragments

[0062] Use the Gel Extraction Kit to purify and recover the above-mentioned target fragments, and follow the operating steps.

[0063] 2. 3′PPM-RACE experiment verifies the interaction between gma-miR396b gene and long-chain acyl-CoA synthetase: ACLS1

[0064] Soybean total RNA extraction

[0065] TRIzon Reagent was used to extract total RNA from soybean pods. The steps were as described in the instructions. The extracted RNA was subjected to electrophoresis and ultra-micro spectrophotometer to analyze RNA quality.

[0066] Add polyA tail

[0067] Poly(A) Polymerase from Bio-Rad Biotechnology (Beijing) Co., Ltd. was used to add polyadenine: poly A to the 3′ end of the extracted RNA.

[0068] Reverse transcription

[0069] The RNA after the above reaction was reverse transcribed using Conway HiFi-MmLV cDNA Kit to obtain cDNA. In the reaction, 3′RACE-RT primer was used for reverse transcription.

[0070] Amplification of the 5′ fragment of the target gene

[0071] The cDNA obtained by reverse transcription was subjected to two rounds of PCR using two characteristic primers and 3′RACE clone-R. The second round template used the 100-fold diluted PCR product of the first round. The PCR reaction conditions are shown in Tables 3, 4 and 5. The target band was subsequently recovered by gel excision, purified and sent to Coomassie for sequencing.

[0072] The first round PCR reaction system is shown in Table 3:

[0073] Table 3 First round PCR reaction system

[0074]

[0075] The second round PCR reaction system is shown in Table 4:

[0076] Table 4 Second round PCR reaction system

[0077]

[0078] The PCR reaction conditions are shown in Table 5:

[0079] Table 5 PCR reaction conditions

[0080]

[0081] The extracted RNA was treated with Poly (A) Polymerase and then reverse transcribed. Using cDNA as a template, two rounds of PCR were performed to obtain the target gene band at 590 bp. Sequence alignment revealed that the gma-miR396b gene specifically recognized the target gene XM_006588497: 984-1005 bp of ACLS1. This cleavage site was consistent with the predicted results, such as Figure 2 .

[0082] 3. Construction of overexpression of gma-miR396b

[0083] Construction of recombinant vector

[0084] The pCAMBI3301 plasmid was linearized using restriction endonucleases NcoI and BstEII, and the digested product was purified and recovered using the GelExtraction Kit. The linearized pCAMBIA3301 vector and the gma-miR396b PCR product were ligated using the Minerva Super Fusion Cloning Kit from Yuheng Bio. The overexpression pCAMBIA3301 vector was double digested with restriction endonucleases NcoI and BstEII. After gel recovery, the electrophoresis detection results of RT-PCR were as follows: Figure 3 The RT-PCR system and reaction conditions were the same as those in Tables 1 and 2.

[0085] Transformation of recombinant plasmid into competent E. coli cells

[0086] In an EP tube, add 5 μL of the above ligation product to 50 μL of competent cells, mix gently and place on ice for 30 minutes. Heat shock in a 42℃ water bath for 45 seconds, and ice water bath for 5 minutes. Add 600 μL of LB medium and culture at 37℃ with shaking for 60 minutes. Pipette 100 mL of bacterial solution and spread on Amp + Solid medium was grown at 37℃ overnight. Single clone colonies were picked, and after liquid shaking culture, they were mixed with 50% glycerol in a 1:1 volume fraction and stored at -80℃. After sequencing, the gma-miR396b overexpression vector plasmid pCAMBIA3301-miR396b was successfully obtained.

[0087] Transformation of recombinant plasmid into Agrobacterium competent cells

[0088] The overexpression vector plasmid pCAMBIA3301-miR396b was transformed into Agrobacterium by freeze-thaw method. The specific steps are as follows: 5pμL of purified plasmid was transferred to 100μL of EHA105 and K599 competent cells respectively, and mixed in an EP tube; the EP tube was placed in an ice bath for 5 minutes, quickly placed in liquid nitrogen for freezing for 5 minutes, and then placed in a 37°C water bath for 5 minutes; 600μL of YEB medium was added to the EP tube, and cultured at 28°C and 100rpm for 4 hours; centrifuged at 4000rpm for 5 minutes, part of the supernatant was removed, and the bacteria were resuspended with the remaining approximately 100μL of liquid, and spread on Kan's YEB solid plate; incubated in a constant temperature incubator at 28°C for 24 hours; a single clone colony was picked and placed in 500μL of YEB liquid culture medium, and liquid shaking cultured overnight; the bacterial solution identified as positive by PCR was stored at -80°C using a volume fraction of 50% glycerol: YEB liquid culture medium 1:1 for subsequent experiments.

[0089] 4. Obtaining gma-miR396b transgenic Arabidopsis plants

[0090] The present invention obtains a transgenic sequence plant, and the method for obtaining the plant comprises the following steps:

[0091] The Arabidopsis thaliana used in the experiment was the Columbia wild-type Arabidopsis thaliana preserved in the laboratory.

[0092] Inflorescence infection method for transformation of Arabidopsis thaliana

[0093] Arabidopsis seeds were vernalized at 4℃ for 48h, nutrient soil and vermiculite were mixed at a ratio of 2:8 and then sterilized by high pressure. The soil was placed in a small pot and placed in a tray. Water was placed in the tray to soak the soil and then Arabidopsis seeds were sown. The culture conditions were 23℃ 16h light / 8h dark. When the inflorescence of Arabidopsis was more after bolting, the opened buds were removed and the bacterial solution was resuspended with the prepared infection solution to an OD value of 0.9. The inflorescence was infective with the Agrobacterium infection solution of OD=0.6. The formula of the infection solution was: 2.2g MS powder, 50g sucrose, 1mL B 5 Vitamins, 100mg / μL 6BA 10μL, 1mol / L NaOH 90μL, surfactant SilwetL-77 200μL. Soak the inflorescence of Arabidopsis in a beaker containing the infection solution. Seal the infected plants, place them in a dark environment at 28℃ for 24 hours, and then culture them in an artificial climate chamber.

[0094] Among them, 1mLB 5 The vitamin formula composition is: 100 mg / mL inositol, 1 mg / mL niacin, 1 mg / mL pyridoxine hydrochloride, and 10 mg / mL thiamine hydrochloride.

[0095] Screening and identification of transgenic Arabidopsis

[0096] Identification and preliminary screening of transgenic Arabidopsis thaliana Use Basta solution at a concentration of 1.5:1000 to spray the seedlings at the four-leaf stage, spray once every 2 days, and after some Arabidopsis seedlings turn yellow, transplant the Arabidopsis thaliana in normal growth state into new small pots. After the seedlings in the pots bolt, extract the rosette leaf genome, use 3301-GUS universal primers to use the genome as a template for PCR, and gel excision and recovery sequencing are performed when the size of the electrophoresis band meets the target sequence. The PCR program and system are the same as Tables 1 and 2, and the rosette leaf genome is used as the template.

[0097] p3301-GUS-universal-F: 5'-TTTCATTTGGAGAGAACACG-3'; p3301-GUS-universal-R: 5'-GGCAACAGGATTCAATCTTA-3'.

[0098] 5. Overexpression of pCAMBIA3301-miR396b gene sequence affects the expression of key fatty acid-related enzyme genes

[0099] The relative expression of key enzyme genes related to fatty acid synthesis, FATB: acyl-ACP thioesterase B, FAD2: fatty acid dehydrogenase, LPAT2: lysophosphatidic acid acyltransferase 2, and LACS8: long-chain acyl-CoA synthetase, in transgenic Arabidopsis and wild-type Arabidopsis was detected by qRT-PCR. The qRT-PCR reaction system is shown in Table 6:

[0100] Table 6 PCR reaction conditions

[0101]

[0102] Relative gene expression values ​​were calculated by 2 -ΔΔCT The qRT-PCR reaction procedure is shown in Table 7:

[0103] Table 7 qRT-PCR reaction procedure

[0104]

[0105] Based on the qRT-PCR primer design principles, Primer 6.0 software was used to design specific primers. The primers were AtLACS8-qF: 5'-ATGGAAGTTGGTTTGGTGCC-3';

[0106] AtLACS8-qR: 5'-CGTTGCGAATCAGGAGACAG-3';

[0107] AtFATB-qF: 5'-CAAGGCCATCCTGAACAATTC-3';

[0108] AtFATB-qR: 5'-TTTTCTTAGCGGCTGAGAAACA-3';

[0109] AtFAD2-qF: 5'-ATGGGTGCAGGTGGAAGAATGCCGG-3';

[0110] AtFAD2-qR: 5'-CCGGCATTCTTCCACCTGCACCCAT-3';

[0111] AtLPAT2-qF: 5'-ATATCGGCGCTTGGTCTAGG-3';

[0112] AtLPAT2-qR: 5'-TGGTGATTGTCTTTTGGCTTG-3'.

[0113] qRT-PCR detection of T 2 The expression levels of key enzyme genes in the gma-miR396b overexpressing plants and wild-type plants were compared. The reaction system and reaction procedure were the same as above.

[0114] Results Figure 4 , Figure 4 A in the figure is the relative expression level of FAD2: fatty acid dehydrogenase; Figure 4 B in it is FATB: relative expression level of fatty acyl-ACP thioesterase B; Figure 4C in the figure is LACS8; relative expression level of long-chain acyl-CoA synthetase; Figure 4 D in the figure is the relative expression level of LPAT2: lysophosphatidic acid acyltransferase 2.

[0115] Depend on Figure 4 It can be seen that compared with the wild type, the expression levels of AtFATB, AtFAD2, AtLPAT2 and AtLACS8 genes in the overexpression positive plants were higher than those in the wild type.

[0116] The above results indicated that the gma-miR396b gene may directly or indirectly affect the expression of AtFATB, AtFAD2, AtLPAT2 and AtLACS8, and then confirmed that the gma-miR396b gene may be involved in the fatty acid synthesis metabolism of Arabidopsis thaliana.

[0117] 6. Overexpression of the gma-miR396b gene reduced oleic acid content

[0118] The T was tested by GB 5009.168-2016S National Food Safety Standard Determination of Fatty Acids in Food (Third Method) 2 Fatty acid contents in seeds of plants overexpressing gma-miR396b and seeds of wild-type Arabidopsis plants.

[0119] Results Figure 5 , Figure 5 A in the figure is the detection map of wild-type Arabidopsis; Figure 5 B in the figure is the detection map of Arabidopsis thaliana overexpressing the gma-miR396b gene sequence.

[0120] Depend on Figure 5 It can be seen that the oleic acid content in plants overexpressing the gma-miR396b gene was significantly lower than that in the control, and the oleic acid content in the grains overexpressing gma-miR396 was 13.793g / 100g, which was a decrease of 14.2% compared with the wild type.

[0121] The above results indicate that the gma-miR396b gene may be involved in the regulation of fatty acids in Arabidopsis thaliana.

[0122] In summary, a deep understanding of the mechanism of the effect of gma-miR396b transgenic on the fatty acid content of Arabidopsis seeds provides a new perspective for a deeper understanding of the regulatory mechanism of plant fatty acid metabolism, and also provides new ideas and methods for plant breeding and bioengineering. The present invention has found through research that the miRNA396b gene can participate in the biosynthesis of soybean fatty acids, and by regulating the expression of the gma-miR396b gene in plants, it is used to cultivate new plant varieties with regulated fatty acid content, providing new ideas for soybean molecular breeding improvement and increasing fatty acid content.

[0123] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0124] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. gma-miR396b The application of a gene in regulating plant fatty acid synthesis is characterized in that: Said gma-miR396b The nucleotide sequence of the gene is shown in SEQ ID NO.1; Overexpression of the gma-miR396b The gene can reduce the content of oleic acid and increase the content of linolenic acid and eicosapentaenoic acid; Using the gma-miR396b Gene constructs an expression vector, and transfers the expression vector into plants to regulate the fatty acid content in the plants; the expression vector is obtained by inserting the gma-miR396b gene into the Nco I and BstE II restriction sites of the pCAMBI3301 vector; The plants are soybean and Arabidopsis thaliana; The gma-miR396b The gene was overexpressed in Arabidopsis to obtain gma-miR396b transgenic Arabidopsis thaliana.

2. according to claim 1 gma-miR396b The application of a gene in regulating plant fatty acid synthesis is characterized in that: Amplify the gma-miR396b Gene primers include gma-miR396b Seamless-F and gma-miR396b Seamless-R; in, gma-miR396b The nucleotide sequence of seamless-F is as shown in SEQ ID NO.3; gma-miR396b The nucleotide sequence of seamless-R is shown in SEQ ID NO.

4.

3. According to claim 1 gma-miR396b The application of a gene in regulating plant fatty acid synthesis is characterized in that: contain gma-miR396b The method for constructing a gene expression vector comprises the following steps: The correct sequencing gma-miR396b The plasmid was used as a template for amplification; The obtained amplified product was double-digested and Nco I and BnE II double-digested pCAMBI3301 vector was connected and transformed, and the correct positive clone plasmid was extracted and verified, which was the expression vector pCAMBIA3301-miR396b.

4. according to claim 3 gma-miR396b The application of a gene in regulating plant fatty acid synthesis is characterized in that: The steps for amplification using pCAMBIA3301-miR396b plasmid as template are as follows: Using pCAMBIA3301-miR396b plasmid as template, gma-miR396b Seamless-F and gma-miR396b Seamless-R was used as a primer pair for amplification; wherein, gma-miR396b The nucleotide sequence of seamless-F is as shown in SEQ ID NO.3; gma-miR396b The nucleotide sequence of seamless-R is shown in SEQ ID NO.4.

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