A paeonia lactiflora pldgat2 gene and its use in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds

Abstract

The application discloses a paeonia lactiflora PlDGAT2 gene and application thereof in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds, and belongs to the technical field of plant biotechnology, wherein the nucleotide sequence of the paeonia lactiflora PlDGAT2 gene is shown as SEQ ID NO. 1. The paeonia lactiflora PlDGAT2 gene can increase the total fatty acid content in the plant seeds after being introduced into the plant seeds, and meanwhile, the proportion of each component of the fatty acid is changed. The application provides a new idea for creating germplasm with strong ability of regulating fatty acid accumulation in plant seeds.

Description

Technical Field

[0001] This invention relates to the peony PLDGAT2 gene and its application in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds, belonging to plant biotechnology. Background Technology

[0002] Peonies are renowned in China for their significant ornamental value. In recent years, with the identification of the related peony (Paeonia lactiflora) as a new type of resource food, the oil-producing value of peonies has also begun to attract attention. In the agronomic traits of oil crops, seed oil content and fatty acid composition are important research hotspots. Effectively increasing seed fat content is currently one of the main goals of breeding various oil crops. In traditional breeding methods, the development of new varieties usually requires many years of selection and hybridization, resulting in slow progress. However, biotechnology can accelerate the breeding process of varieties with high seed oil content and high-quality fatty acid composition. By using biotechnology to alter the metabolic pathways of crop oil synthesis, the content of unsaturated fatty acids in oils can be increased, thus creating traits with high unsaturated fatty acid content.

[0003] Members of the diacylglycerol acyltransferase (DGAT) gene family were first characterized more than two decades ago. These genes encode enzymes that play a central role in plant oil production, metabolic regulation, and stress responses. They are considered key enzymes in the conversion of diacylglycerol (DAG) to triacylglycerol (TAG), leading to their extensive study in a range of plant species. They are also the only rate-limiting enzymes involved in the Kennedy pathway, which regulates de novo TAG biosynthesis.

[0004] Currently, four classes of DGAT genes have been identified in plants, including DGAT1, DGAT2, DGAT3, and WSD / DGAT. These genes exhibit plant-specific and tissue-specific characteristics, and their roles in lipid synthesis and accumulation differ. Numerous studies have shown that DGAT plays a crucial role in TAG synthesis. In plants, the different genes encoding DGAT determine its varying enzyme activities, leading to different roles in TAG synthesis and accumulation across different tissues. DGAT2 typically ranges in length from 315 to 373 amino acids, and it also exhibits substrate preference for different fatty acids in different species. During castor bean seed development, DGAT2 is expressed at higher levels than DGAT1, contributing to the accumulation of ricinoleic acid (18:1). In diatoms, DGAT2 expression in yeast systems increases palmitic acid (16:0) and oleic acid (18:1). DGAT2 is primarily expressed in late-maturing soybean seeds, potentially participating in lipid accumulation in late-maturing seeds. Peony DGAT2 exhibits a unique substrate preference in the accumulation of linolenic acid (18:3). However, no reports have been found of DGAT2 in the accumulation of fatty acids in peony seeds. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a peony PLDGAT2 gene and its application in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds.

[0006] Technical solution: The present invention provides a peony PLDGAT2 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a protein encoded by the above-mentioned gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides amplification primers for cloning the full-length cDNA sequence of the peony PLDGAT2 gene, the sequences of which are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0009] This invention also provides the application of expression cassettes, recombinant vectors, recombinant cells or recombinant strains containing the above-mentioned peony PLDGAT2 gene in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds.

[0010] Furthermore, the regulation of fatty acid composition in plant seeds refers to increasing the content of palmitic acid and / or α-linolenic acid in the seeds.

[0011] Furthermore, the plant includes tobacco.

[0012] Furthermore, the recombinant vector is obtained by introducing the ORF sequence of the peony PLDGAT2 gene into a plant binary expression vector.

[0013] Furthermore, the primer sequences used to amplify the ORF sequence of the peony PLDGAT2 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] The present invention also provides a method for identifying seeds of plants with high fatty acids, comprising the following steps:

[0015] 1) To identify whether the seeds of the plant contain the peony PLDGAT2 protein with the amino acid sequence shown in SEQ ID NO.2;

[0016] 2) Identify whether the plant seeds contain the peony PLDGAT2 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0017] Furthermore, the primer sequences used for PCR identification are shown in SEQ ID NO.3 and SEQ ID NO.4; the primer sequences used for qRT-PCR identification are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0018] Beneficial effects: Compared with existing technologies, this invention has the following significant advantages: the peony PLDGAT2 gene of this invention, after being transferred into plant seeds, can increase the total fatty acid content and simultaneously alter the proportions of various fatty acid components. This provides a new approach for creating germplasm with strong ability to regulate fatty acid accumulation in plant seeds. Attached Figure Description

[0019] Figure 1 PCR results of the full-length cDNA of the peony PLDGAT2 gene; where M: DL 2000 marker; 1: PCR amplification product.

[0020] Figure 2 Phylogenetic analysis of the amino acid sequence of the peony PLDGAT2 gene with the DGAT family in Arabidopsis and rice. Note: At: Arabidopsis; Os: rice.

[0021] Figure 3 Amino acid sequence alignment of PLDGAT2 and its homologous DGAT2 protein; Note: At: Arabidopsis thaliana; Os: rice

[0022] Figure 4 Subcellular localization analysis of PLDGAT2; Note: Scale bar size is 25 μm;

[0023] Figure 5 Identification of tobacco plants transgenic with the PLDGAT2 gene based on RT-PCR detection: The left figure shows the PCR results of the NtActin primer; the middle figure shows the PCR results of the PLDGAT2 primer; and the right figure shows the PCR results of the P1301-PlDGAT2 primer.

[0024] Figure 6 Identification of PLDGAT2 gene-transgenic tobacco plants based on qRT-PCR detection: Different lowercase letters indicate significant differences (P<0.05).

[0025] Figure 7 : Total fatty acid content in tobacco seeds overexpressing the PLDGAT2 gene. Different lowercase letters indicate significant differences (P<0.05).

[0026] Figure 8 Fatty acid composition analysis in tobacco seeds overexpressing the PLDGAT2 gene. Different lowercase letters indicate significant differences (P<0.05). Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0028] Unless otherwise specified, the experimental methods in the following examples were performed according to conventional procedures. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1: Cloning of the full-length cDNA sequence of the peony PLDGAT2 gene

[0030] Obtaining the full-length sequence of the PLDGAT2 gene: The leaves of the peony variety 'Hangshao' were used as material, and total RNA was extracted using the MiniBESTPlant RNAExtraction Kit (TaKaRa).

[0031] cDNA was produced by reverse transcription using the PrimeScript RT Reagent Kit (TaKaRa). The reverse transcription system consisted of: 1 μL RNA, 1 μL Oligo(dT)18, 1 μL dNTP Mixture (10 mM each), 2 μL 5× PrimeScript Buffer, 0.25 μL RNase Inhibitor, 0.25 μL PrimeScript RTase, and 4.5 μL RNase FreeddH2O. The reverse transcription program was: 42℃ for 60 min, followed by an extension at 70℃ for 15 min. Following this, PCR amplification was performed. The PCR amplification system consisted of: 2 μL cDNA, 2 μL dNTP Mixture (10 mM each), and 2.5 μL 10× Vazyme Lamb Buffer (Mg). 2+ The reaction mixture consisted of 1 μL Forward Primer (5'-ATCCCACTAACGAAGC-3' (SEQ ID NO. 9)), 1 μL Reverse Primer (5'-GCCCACCACAATTTAA-3' (SEQ ID NO. 10)), 0.25 μL Vazyme LAmp DNA Polymerase (5 U / μL), and 16.3 μL RNase-Free ddH2O. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 35 cycles; and 72℃ extension for 10 min. The products were detected by 1% agarose gel electrophoresis. The results are shown in the figure below. Figure 1 .

[0032] Sequencing yielded the full-length cDNA sequence of the gene PLDGAT2 (SEQ ID NO).1):ATGGAGAACGATTCG GGAATGTCTCAGCCGTCGGCCGTGGATAAACCGACGGTATTCAAGGGAAAAGAAGAGTCCACAATACAGACAATTGCAGCTTTGGCAGTATGGCTTGGCGCCATCCATTTCAATGTCGCTGTAGTCCTCTTCGCGATATTCTTCCTTCCTCTTCGCAACGCCTTCGCGGTGTTTGGATTGCTTATATTTTTAATGGTTATACCAATTGACGATAGCAGCAAAGTAGGTCGAAGGTTATCCAGGTATATATGTAAGCATGCCTGTGGTTATTTTCCGGTGACTCTACATGTGGAGGATATAGAGGCCTTTGATCCAAATCGTGCATATGTCTTTGGTTATGAGCCCCATTCAGTTTTGCCAATTGGTGTCATCGCACTTGCCAACCTTACGGGTTTCATGCCTCTCCCAAAAATAAAGGTCCTCGCAAGTAGTGCTGTGTTCTACACACCATTCTTGAGGCATATATGGACATGGTTGGGTCTTGCACCTGCAACAAAGGAAGGTTTTATTTCCCTTTTGTCAGCTGGTTATAGTTGCCTCGTAATACCTGGTGGAGTGCAAGAAACATTTCATATGGAGCATGGTTCTGAGAATGTTTTCCTGAAGACGAGAAGAGGATTTGTTCGTATTGCCATGGAGATGGGCCTACCCCTAGTTCCAGTTTTCTGTTTTGGCCAGTCACGTGTCTACAAGTGGTGGAAGCCTGGCGGGGAACTGTATTTGCAATTTTCTAGAGCTATCAAATTCACTCCAATTTTTTTTTGGGGAAAATTTGGATCTCCAATCCCCTTTCGACATCCAATGCATGTGGTTGTAGGAAGACCCATTGAGCTTAAGAAAAATCCACAACCCACTATGGAAGAGGTGATTCAAGTACACGGTCAGTTCGTTGAAGCACTAAAGAATCTGTTTGAAAAGCACAAATCACGGGTTGGCCATGCTGATCTTGAATTAAAAATATTGTGA。.

[0033] Amino acid sequence deduced from the cDNA sequence of gene PlDGAT2 (SEQ ID NO. 2): MENDSG MSQPSAVDKPTVFKGKEESTIQTIAALAVWLGAIHFNVAVVLFAIFFLPLRNAFAVFGLLIFLMVIPIDDSSKVGRRLSRYICKHACGYFPVTLHVEDIEAFDPNRAYVFGYEPHSVLPIGVIALANLTGFMPLPKIKVLASSAVFYTPFLRHIWTWLGL APATKEGFISLSAGYSCLVIPGGVQETFHMEHGSENVFLKTRRGFVRIAMEMGLPLVPVFCFGQSRVYKWWKPGGELYLQFSRAIKFTPIFFWGKFGSPIPFRHPMHVVVGRPIELKKNPQPTMEEVIQVHGQFVEALKNLFEKHKSRVGHADLELKIL.

[0034] Example 2: Comparison of the amino acid sequence of peony PLDGAT2 protein with that of Arabidopsis thaliana and rice DGAT family.

[0035] The amino acid sequences of the Arabidopsis DGAT family were downloaded from the databases of The Arabidopsis Information Resource (TAIR) (https: / / www.arabidopsis.org / ) and the National Rice Data Center (https: / / www.ricedata.cn / ). These sequences, along with the amino acid sequences of the peony PLDGAT2 protein, were represented in FASTA format. A phylogenetic tree was then constructed using MEGA 7.0 software with the Neighbor-Joining algorithm and 1000 self-reviews. The most homologous amino acid sequences were observed, showing that it clustered with Arabidopsis AtDGAT2. Figure 2 Further analysis of the amino acid sequences of DGAT2, a homologous protein in Arabidopsis and rice that clusters with PLDGAT2 in the same phylogenetic branch on the family tree, revealed that they all contain a conserved LPLAT superfamily structure, indicating that they belong to the same gene family. Figure 3 ).

[0036] Example 3: Expression of the peony PLDGAT2 gene overexpression vector in tobacco

[0037] Construction of the overexpression vector for the peony PLDGAT2 gene: Primers P1301-PlDGAT2 containing BamHI and KpnI restriction sites were designed for amplifying the PLDGAT2 sequence (forward primer PLDGAT2-F: 5'-aaggttaccgaattctctagaTTCAAGATCAGCATGGCCAAC-3' (SEQ ID NO.3), downstream primer PLDGAT2-R: 5'-cgtgagctcggtaccggatccTCTCCAATCCCCTTTCGACA-3' (SEQ ID NO.4)). PCR amplification system: 12.5 μL 2×Phanta FlashMaster Mix (Vazyme), 1 μL Forward Primer, 1 μL Reverse Primer, 2 μL DNA template (peony leaf DNA extracted using the NuClean PlantGenomic DNA Kit (CWBIO) was used as the template), 8.5 μL ddH2O. Reaction procedure: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 52℃ annealing for 5 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ extension for 1 min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the large fragment of PLDGAT2 containing the restriction enzyme site (ORF sequence of cDNA) was recovered using the TSP601-DNA gel recovery kit (Tsingke). The binary expression vector pCAMBIA1301 plasmid (laboratory stock) was double-digested with BamHI and KpnI. The reaction system was: 2.0 μL 10×CutSmart Buffer, 7 μL pCAMBIA1301 plasmid, 0.4 μL BamHI (20000 U / mL), 0.4 μL KpnI (20000 U / mL), 10.2 μL ddH2O; reaction at 37℃ for 1 h. The double enzyme digestion products were analyzed by agarose gel electrophoresis, and the large fragment of plasmid pCAMBIA1301 was recovered and purified using the TSP601-DNA gel recovery kit (Tsingke).The two recovered products were ligated using the homologous recombination method with the plus Onestep PCR Cloning Kit (Novoprotein). The reaction system consisted of: 4.0 μL 5× reaction buffer, 1.0 μL plus recombinase, 9 μL pCAMBIA1301 fragment, 6 μL P1DGAT2 fragment, and 7.0 μL ddH2O. After ligation in a 50°C metal bath for 15 min, the cells were cooled on ice. 5 μL of the ligation product was transformed into Trelief™ 5α competent cells (Tsingke) and then cultured overnight at 37°C on LB plates (containing 50 mg / L Kana). Positive clones were picked and expanded, and the plasmid pCAMBIA1301-PlDGAT2 was extracted. Double enzyme digestion and sequencing were then performed for verification until the pCAMBIA1301-PlDGAT2 overexpression vector was successfully constructed.

[0038] Transformation of tobacco with the peony PLDGAT2 gene overexpression vector: 5 μL of the pCAMBIA1301-PlDGAT2 overexpression vector plasmid was transformed into GV3101 (pSoup-p19) competent cells (TOLOBIO). The cells were then cultured on YEB plates (containing 50 mg / L Rif and 50 mg / L Kan) at 28°C for 2 days. Positive clones were picked and cultured overnight at 28°C and 200 rpm in YEB liquid medium (containing 50 mg / L Rif and 50 mg / L Kan). 2 mL of the culture was added to 50 mL of liquid YEB containing the same antibiotics (50 mg / L Rif and 50 mg / L Kan), and cultured under the same conditions until OD (Organic Overflow Rate). 600=0.3-0.4. Pour the shaken bacteria into a 50mL centrifuge tube, centrifuge at 5000rpm for 10min at room temperature, and discard the supernatant. First, add 100μL of acetylsylgenone (20mg / mL) to a sterilized small Erlenmeyer flask, then add 5mL of MS0 (MS liquid basal medium, without agar and sucrose) to the centrifuge tube to dissolve the bacteria, mix well with a pipette, pour into the small Erlenmeyer flask containing acetylsylgenone, and then add MS0 to make up to 50mL. Add 50 mL of MS0 (MS liquid basal medium, without agar and sucrose) to another sterile Erlenmeyer flask. Take sterile tobacco seedling leaves, cut them into small pieces (about 1 cm × 1 cm), and cut 100-150 pieces in total. Place the leaves into the Erlenmeyer flask containing only 50 mL of MS0, gently shake for 1-3 minutes, and then pour the leaves into a beaker lined with gauze. Add the filtered leaves to the previously prepared Erlenmeyer flask containing bacterial cells and incubate for 8 minutes, gently shaking continuously during incubation. After incubation, filter out the bacterial solution, remove the leaves, and blot off excess bacterial solution from the leaf surface with sterile filter paper. Inoculate the leaves into co-culture medium [MS + 3.0 mg / L 6-benzylaminopurine (6-BA) + 0.1 mg / L 1-Naphthaleneacetic acid (NAA) + 30 g / L sucrose + 6.66% agar] and incubate in the dark for 3 days. After co-culture, transfer the leaves to resistant bud selection and differentiation medium [MS + 3.0 mg / L 6-BA]. Selective culture was conducted using a medium containing 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 6.66% agar + 100 mg / L Carbenicillin (Cb) + 25 mg / L Hygromycin (Hyg), with subculture every two weeks until shoot differentiation occurred. When adventitious shoots reached 2 cm or more, they were cut off and transferred to rooting selection medium [1 / 2 MS + 0.3 mg / L 2-Iodosobenzoic acid (IBA) + 30 g / L sucrose + 6.66% agar + 50 mg / L Cb + 8 mg / L Hyg] for rooting selection. After 4-6 months of culture, PLDGAT2 gene-transgenic tobacco could be obtained.

[0039] Example 4: Subcellular localization analysis of PLDGAT2

[0040] To investigate the subcellular localization of PLDGAT2, an eGFP nucleic acid sequence was introduced into the recombinant vector pCAMBIA2300-PlDGAT2. The recombinant vector pCAMBIA2300-PlDGAT2-eGFP was then transformed into Agrobacterium GV3101 (pSoup-p19) (Shanghai Weidi Biotechnology Co., Ltd.). Agrobacterium GV3101 (pSoup-p19) carrying the PLDGAT2-GFP and those carrying only the GFP sequence were mixed with Agrobacterium GV3101 (pSoup-p19) carrying the pCAMBIA1300-mcherry vector. After standing in the dark for 1 hour, approximately 2-3 ml was injected into the underside of tobacco leaves using a disposable syringe. After 3 days of culture, the expression of GFP and RFP (vectors with endoplasmic reticulum markers) was observed using a confocal microscope. The results are as follows: Figure 4 The results showed that PLDGAT2-GFP was localized in the endoplasmic reticulum in tobacco leaves, consistent with the cellular localization of control GFP and RFP (a vector with an endoplasmic reticulum marker), indicating that PLDGAT2 plays a role in the endoplasmic reticulum. This also provides a locational basis for PLDGAT2 to perform its acyltransferase transport function in the endoplasmic reticulum.

[0041] Example 5: Identification of tobacco plants transfected with the peony PLDGAT2 gene

[0042] PCR identification: DNA was extracted from leaves of wild-type tobacco and three transgenic tobacco plants obtained in Example 3 using the NuClean Plant Genomic DNA Kit (CWBIO). Based on this, the tobacco NtActin (AB158612) gene was used as an internal control (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.5), Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.6)). Simultaneously, PCR amplification was performed using Hyg primers (Forward Primer: 5'-CTGCCCGCTGTTCTACAACCGG-3' (SEQ ID NO.11), Reverse Primer: 5'-GGAGCATATACGCCCGGAGTC-3' (SEQ ID NO.12)) and primers P1301-PlDGAT2 (SEQ ID NO.3, SEQ ID NO.4) constructed from the PLDGAT2 gene overexpression vector. Reaction system: 12.5 μL 2×RapidTaq Master Mix (Vazyme), 1 μL Forward Primer, 1 μL Reverse Primer, 2 μL DNA template, 8.5 μL ddH2O. Reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 52℃ annealing for 15 s, 72℃ extension for 5 s, for a total of 35 cycles; 72℃ extension for 5 min. After the reaction, the PCR reaction solution was analyzed by gel electrophoresis. Figure 5 It can be seen that a single, bright NtActin band was detected in both wild-type tobacco and tobacco transgenic with the peony PLDGAT2 gene. However, regarding the amplified Hyg band, a single, bright, and clear band was detected only in tobacco transgenic with the peony PLDGAT2 gene, and not in wild-type tobacco. At the same time, when amplifying the PLDGAT2 gene, a single, bright, and clear band was detected only in tobacco transgenic with the peony PLDGAT2 gene.

[0043] qRT-PCR identification: Total RNA was extracted from leaves of wild-type tobacco and three transgenic tobacco plants obtained in Example 3 using the MiniBEST Plant RNA Extraction Kit (TaKaRa). The total RNA was reverse transcribed into cDNA using the HiScript III RTSuperMix for qPCR (+gDNA wiper) (Vazyme) kit. The reaction mixture consisted of 1.0 μL RNA, 4.0 μL 4×gDNA wiper Mix, and 11.0 μL RNase-free dH2O. The reaction conditions were 42℃ for 2 min. After the reaction, 4.0 μL 5×HiScript III qRT SuperMix was added to the reaction mixture from the first step. The reaction conditions were 37℃ for 15 min followed by 85℃ for 5 s. The cDNA obtained from the reverse transcription was then detected by qRT-PCR using the SYBR qPCR SuperMix Plus (Novoprotein) kit. Based on this, using the tobacco NtActin (AB158612) gene as an internal reference (SEQ ID NO.5, SEQ ID NO.6), and designing specific primers for the PLDGAT2 gene (Forward Primer: 5'-TACAAGTGGTGGAAGCC-3' (SEQ ID NO.7), Reverse Primer: 5'-CATTGGATGTCGAAAGG-3' (SEQ ID NO.8)) for qRT-PCR detection. The reaction system consisted of 2 μL cDNA, 12.5 μL LSYBR qPCR SuperMixPlus, 1 μL Forward Primer, 1 μL Reverse Primer, and 8.5 μL ddH2O. The reaction program was: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles; and 72℃ extension for 10 min. After the reaction, 2... -△△ The relative gene expression level was analyzed using the CT method. qRT-PCR identification results showed that PLDGAT2 had a significantly high expression level in transgenic tobacco. Figure 6 ).

[0044] Example 6: Identification of the ability of the peony PLDGAT2 gene to regulate fatty acid composition in plant seeds.

[0045] The transgenic tobacco obtained in Example 3 was transplanted and the plants were cultured until seed harvest. After drying the tobacco seeds in an oven at 65℃, fatty acid extraction was performed, followed by GC-MS analysis, and qualitative and quantitative analysis of the relevant fatty acids. The results are as follows: Figure 7The results showed that, compared with wild tobacco control, the total fatty acid content in tobacco seeds overexpressing the PLDGAT2 gene increased by 20%, indicating that overexpression of PLDGAT2 can increase the fatty acid content in tobacco seeds. Furthermore, analysis of the composition of tobacco seeds overexpressing the PLDGAT2 gene yielded the following results: Figure 8 The results showed that, compared with the control, the palmitic acid (C16:0) content in transgenic tobacco seeds OE-L1, OE-L2, and OE-L3 increased by 17.84%, 13.63%, and 15.92%, respectively. Meanwhile, the linolenic acid (C18:3) content in OE-L1, OE-L2, and OE-L3 tobacco seeds increased by 25.48%, 26.58%, and 25.56%, respectively, while the linoleic acid (C18:2) content in OE-L1 and OE-L2 tobacco seeds decreased by 5.90% and 9.01%, respectively. Other fatty acid compositions did not change significantly. Figure 8 ).

[0046] In summary, overexpression of PLDGAT2 can promote the accumulation of palmitic acid (C16:0) and α-linolenic acid (C18:3), and tobacco transgenic with the PLDGAT2 gene has a strong ability to regulate the fatty acid composition in plant seeds, especially to promote the accumulation of α-linolenic acid (18:3).

[0047] In summary, this invention provides a full-length cDNA sequence of the peony PLDGAT2 gene and its application in regulating fatty acid composition in plant seeds. By converting the constructed PLDGAT2 gene overexpression vector into tobacco for expression, a new tobacco germplasm with strong ability to control fatty acid composition in plant seeds was created.

Claims

1. The application of overexpression of the PLDGAT2 protein shown in SEQ ID NO.2 in regulating fatty acid composition in plant seeds, characterized in that, The regulation of fatty acid composition in plant seeds refers to increasing the content of palmitic acid and / or α-linolenic acid in the seeds, with the plant being tobacco.

2. Contains as shown in SEQ ID NO.1 PLDGAT2 The application of gene expression cassettes, recombinant vectors, recombinant cells, or recombinant strains in regulating fatty acid composition in plant seeds, characterized by: The regulation of fatty acid composition in plant seeds refers to increasing the content of palmitic acid and / or α-linolenic acid in the seeds, with the plant being tobacco.

3. The application according to claim 2, characterized in that, The recombinant vector uses peony... PLDGAT2 The ORF sequence of the gene was obtained by introducing it into a plant binary expression vector.

4. The application according to claim 3, characterized in that, Used to amplify peony PLDGAT2 The primer sequences for the gene's ORF sequence are shown in SEQ ID NO.3 and SEQ ID NO.4.

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