Paeonia lactiflora PlDGAT2 gene and application thereof in increasing total fatty acid content and / or regulating fatty acid composition in plant seeds

Abstract

The invention discloses a paeonia lactiflora PlDGAT2 gene and application of the paeonia lactiflora PlDGAT2 gene in increasing the total fatty acid content and / or regulating and controlling the fatty acid composition in plant seeds, and belongs to the plant biotechnology, and the nucleotide sequence of the paeonia lactiflora PlDGAT2 gene is shown as SEQ ID NO.1. After the paeonia lactiflora PlDGAT2 gene is transferred into plant seeds, the total fatty acid content can be increased, and meanwhile the proportion of all components of fatty acid is changed. A new thought is provided for creating germplasm with strong fatty acid accumulation capacity in the plant seeds.

Description

Technical Field

[0001] The invention relates to a 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 Art

[0002] Peony is well known for its important ornamental value. In recent years, as the peony of the same genus was identified as a new resource food, the oil value of peony has also begun to receive attention. In the agronomic traits of oil crops, seed oil content and fatty acid types are important research hotspots. Effectively increasing seed fat content is one of the main goals of oil crop breeding. In traditional breeding methods, breeding new varieties usually requires years of selection and hybridization, and progress is slow. Biotechnology can accelerate the breeding process of varieties with high seed oil content and high-quality fatty acid components. Using biotechnology to change the metabolic pathway of crop oil synthesis can increase the content of unsaturated fatty acids in oils and achieve the creation of high unsaturated fatty acid traits.

[0003] Members of the diacylglycerol acyltransferase (DGATs) gene family were first characterized more than two decades ago. The enzymes encoded by these genes play a central role in plant oil production, metabolic regulation, and stress response and are considered to be key enzymes for 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 enzyme involved in the Kennedy pathway that regulates de novo TAG biosynthesis.

[0004] At present, four types of DGAT genes have been found in plants, including DGAT1, DGAT2, DGAT3 and WSD / DGAT. They are plant-specific and tissue-specific, and play different roles in oil synthesis and accumulation. A large number of studies have shown that DGAT plays a vital role in the synthesis of TAG. In plants, DGAT has different enzyme activities determined by different genes, which makes its role in the synthesis and accumulation of TAG in different tissues of plants different. Usually, the length of DGAT2 is between 315-373 amino acids, and DGAT2 also has substrate preferences for different fatty acids in different species. During the development of castor seeds, DGAT2 is expressed at a higher level than DGAT1, which helps the accumulation of ricinoleic acid (18:1). When DGAT2 in diatoms is expressed in the yeast system, palmitic acid (16:0) and oleic acid (18:1) are increased. DGAT2 is mainly expressed in the late maturity of soybean seeds and may be involved in the oil accumulation of late-maturing seeds. Peony DGAT2 showed a unique substrate preference in the accumulation of linolenic acid (18:3). However, there is no report on the role of DGAT2 in the accumulation of fatty acids in peony seeds. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a peony PlDGAT2 gene and its application in increasing the total fatty acid content and / or regulating the fatty acid composition in plant seeds.

[0006] Technical solution: The present invention provides a peony PlDGAT2 gene, the nucleotide sequence of the peony PlDGAT2 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides a protein encoded by the above gene, the amino acid sequence of the protein is shown as 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 the amplification primers are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0009] The present invention also provides the use of an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the above-mentioned peony PlDGAT2 gene in increasing the total fatty acid content and / or regulating the fatty acid composition in plant seeds.

[0010] Furthermore, regulating the 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 Paeonia lactiflora PlDGAT2 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] The present invention also provides a method for identifying high fatty acid plant seeds, comprising the following steps:

[0015] 1) identifying whether the plant seeds contain the peony PlDGAT2 protein with an amino acid sequence as shown in SEQ ID NO.2;

[0016] 2) Identifying 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 the prior art, the present invention has the following outstanding and significant advantages: the peony PlDGAT2 gene of the present invention can increase the total fatty acid content after being transferred into plant seeds, and at the same time change the ratio of each fatty acid component, providing a new idea for creating germplasm with strong fatty acid accumulation ability in regulating plant seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : PCR results of the full-length cDNA of peony PlDGAT2 gene; where M: DL 2000 marker; 1: PCR amplification product.

[0020] Figure 2 :Analysis of amino acid sequence of peony PlDGAT2 gene and phylogenetic tree of DGAT family in Arabidopsis and rice. Note: At: Arabidopsis; Os: rice

[0021] Figure 3 :Amino acid sequence alignment of PlDGAT2 and homologous DGAT2 proteins; Note: At: Arabidopsis; Os: Rice

[0022] Figure 4 :Subcellular localization analysis of PlDGAT2; Note: The scale bar is 25 μm;

[0023] Figure 5 : Identification of transgenic tobacco plants with the P1DGAT2 gene based on RT-PCR detection: The left picture shows the PCR result of NtActin primers; the middle picture shows the PCR result of P1DGAT2 primers; the right picture shows the PCR result of P1301-P1DGAT2 primers.

[0024] Figure 6 :Identification of transgenic tobacco plants expressing PlDGAT2 gene 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 PlDGAT2 gene. Different lowercase letters indicate significant differences (P<0.05).

[0026] Figure 8 :Analysis of fatty acid composition in tobacco seeds overexpressing PlDGAT2 gene. Different lowercase letters indicate significant differences (P<0.05). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] The experimental methods in the following examples without specifying specific conditions were all carried out according to conventional procedures. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[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: Leaves of the peony variety 'Hang Shao' were selected as materials, and total RNA was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa).

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

[0032] The full-length cDNA sequence of gene PlDGAT2 was obtained by sequencing (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 Alignment of the amino acid sequence of the peony PlDGAT2 protein with the Arabidopsis and rice DGAT families

[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 / ). The amino acid sequences of the Arabidopsis DGAT family and the peony PlDGAT2 protein were expressed in FASTA format, and the evolutionary tree was constructed using MEGA 7.0 software. The algorithm was Neighbor·Joining, and the self-introduction was performed 1000 times. The most homologous amino acid sequences were observed, and the results showed that it was clustered with Arabidopsis AtDGAT2 ( Figure 2 Further analysis of the amino acid sequences of Arabidopsis and rice homologous proteins DGAT2, which are clustered in the same evolutionary branch as PlDGAT2 in the family evolutionary tree, revealed that they both contain a conserved LPLAT superfamily structure, indicating that they belong to the same family of genes ( Figure 3 ).

[0036] Example 3 Expression of Peony PlDGAT2 Gene Overexpression Vector in Tobacco

[0037] Construction of overexpression vector of peony PlDGAT2 gene: Primers P1301-PlDGAT2 containing restriction sites BamH I and Kpn I were designed to amplify the PlDGAT2 sequence (upstream 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 (DNA extracted from peony leaves using NuClean PlantGenomic DNA Kit (CWBIO) as template), 8.5 μL ddH2O. Reaction procedure: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 52℃ annealing for 5s, 72℃ extension for 10s, 35 cycles in total; 72℃ extension for 1min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the TSP601-DNA gel recovery kit (Tsingke) was used to recover the large fragment of PlDGAT2 containing the restriction site (ORF sequence of cDNA). The binary expression vector pCAMBIA1301 plasmid (laboratory storage) was taken and double-digested with BamH I and Kpn I. The reaction system was: 2.0μL 10× CutSmart Buffer, 7μL pCAMBIA1301 plasmid, 0.4μL BamH I (20000U / mL), 0.4μL Kpn I (20000U / mL), 10.2μL ddH2O; 37℃ reaction for 1h. The double-enzyme digestion products were analyzed by agarose gel electrophoresis, and the large fragment of plasmid pCAMBIA1301 was recovered and purified using TSP601-DNA gel recovery kit (Tsingke).The two recovered products were connected by homologous recombination using the plus Onestep PCR Cloning Kit (Novoprotein). The reaction system was: 4.0 μL 5× reaction buffer, 1.0 μL plus recombinase, 9 μL pCAMBIA1301 large fragment, 6 μL pPlDGAT2 large fragment, 7.0 μL ddH2O; after connection in a 50°C metal bath for 15 minutes, the cells were cooled on ice, 5 μL of the connection product was taken to transform TreliefTM5α competent cells (Tsingke), and then cultured on LB plates (containing 50 mg / L Kana) at 37°C overnight, positive single clones were picked for expansion culture, plasmid pCAMBIA1301-PlDGAT2 was extracted, and double enzyme digestion and sequencing were performed for verification until the pCAMBIA1301-PlDGAT2 overexpression vector was successfully constructed.

[0038] Transformation of tobacco with peony PlDGAT2 gene overexpression vector: Take 5 μL of pCAMBIA1301-PlDGAT2 overexpression vector plasmid to transform GV3101 (pSoup-p19) competent cells (TOLOBIO), and then culture on YEB plates (containing Rif50 mg / L and Kana 50 mg / L) at 28°C for 2 days, pick positive single clones in YEB liquid culture medium (containing Rif50 mg / L and Kan 50 mg / L), and culture overnight at 28°C and 200 rpm. Take 2 mL of the shaken bacterial solution and add it to 50 mL of liquid YEB (containing Rif50 mg / L and Kan 50 mg / L) containing the same antibiotics, and culture under the same conditions until OD 600=0.3-0.4. Pour the shaken bacteria into a 50mL centrifuge tube, centrifuge at room temperature at 5000rpm for 10min, and discard the supernatant for later use. First, add 100μL of acetosyringone (20mg / mL) to a sterilized small triangle bottle, add 5mL of MS0 (MS liquid basic medium, without agar and sucrose) to the centrifuge tube to dissolve the bacteria, beat with a gun, pour into the small triangle bottle containing acetosyringone, and then add MS0 to make up to 50mL. Add 50mL MS0 (MS liquid basic medium, without agar and sucrose) to another sterilized small triangular flask, take the sterile seedling leaves of tobacco, cut them into small pieces (about 1cm×1cm), cut 100-150 pieces in total, put the leaves into the small triangular flask containing only 50mL MS0, shake gently for 1-3 minutes, pour the leaves into a beaker covered with gauze, take the filtered leaves and add them to the small triangular flask containing bacteria prepared before, infect for 8min, and shake gently during the infection; after the infection is completed, filter out the bacterial solution, take out the leaves, use sterile filter paper to absorb the excess bacterial solution on the surface of the leaves, inoculate them in the co-culture medium [MS+3.0mg / L 6-benzylaminopurine (6-BA)+0.1mg / L 1-Naphthaleneacetic acid (NAA)+30g / L sucrose+6.66% agar], and culture them in the dark for 3d; after the co-culture is completed, transfer to the resistance bud screening differentiation medium [MS+3.0mg / L 6-BA+0.1mg / L NAA+30g / L sucrose+6.66% agar+100mg / L Carbenicillin(Cb)+25mg / L Hygromycin(Hyg)] for selection culture, subculture once every two weeks until differentiation and budding; when the adventitious buds reach more than 2cm, cut off the adventitious buds and transfer to rooting screening medium [1 / 2MS+0.3mg / L 2-Iodosobenzoic acid(IBA)+30g / L sucrose+6.66% agar+50mg / L Cb+8mg / L Hyg] for rooting screening. After 4-6 months of culture, transgenic tobacco with PlDGAT2 gene can be obtained.

[0039] Example 4 Subcellular localization analysis of P1DGAT2

[0040] In order to study the subcellular localization of PlDGAT2, the eGFP nucleic acid sequence was introduced into the recombinant vector pCAMBIA2300-PlDGAT2, and the recombinant vector pCAMBIA2300-PlDGAT2-eGFP was transferred into Agrobacterium GV3101 (pSoup-p19) (Shanghai Weidi Biotechnology Co., Ltd.). Agrobacterium GV3101 (pSoup-p19) carrying PlDGAT2-GFP and only carrying GFP sequence were mixed with Agrobacterium GV3101 (pSoup-p19) carrying pCAMBIA1300-mcherry vector, respectively, and left to stand in the dark for 1 hour, and then about 2-3 ml was injected into the back of tobacco leaves with a disposable syringe. After culturing for 3 days, the expression of GFP and RFP (vector with endoplasmic reticulum marker) was observed using a confocal microscope. The results are shown in the figure. Figure 4 The results showed that PlDGAT2-GFP was localized in the endoplasmic reticulum in tobacco leaves, which was consistent with the cellular localization of the control GFP and RFP (vector with an endoplasmic reticulum marker), indicating that PlDGAT2 played a role in the endoplasmic reticulum, which also provided a positional basis for the transport function of PlDGAT2 as an acyltransferase in the endoplasmic reticulum.

[0041] Example 5 Identification of tobacco plants transformed with the peony PlDGAT2 gene

[0042] PCR identification: NuClean Plant Genomic DNA Kit (CWBIO) was used to extract DNA from wild-type tobacco and three transgenic tobacco leaves obtained in Example 3. On this basis, PCR amplification was performed using tobacco NtActin (AB158612) gene as an internal reference (Forward Primer: 5'-TCCTCATGCAATTCTTCG-3' (SEQ ID NO.5), Reverse Primer: 5'-ACCTGCCCATCTGGTAAC-3' (SEQ ID NO.6)), and Hyg primers (Forward Primer: 5'-CTGCCCGCTGTTCTACAACCGG-3' (SEQ ID NO.11), Reverse Primer: 5'-GGAGCATATACGCCCGGAGTC-3' (SEQ ID NO.12)) and primers P1301-P1DGAT2 (SEQ ID NO.3, SEQ ID NO.4) for constructing the overexpression vector of the P1DGAT2 gene. Reaction system: 12.5μL 2×RapidTaq Master Mix (Vazyme), 1μL Forward Primer, 1μL Reverse Primer, 2μL DNA template, 8.5μL ddH2O. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 52℃ annealing for 15s, 72℃ extension for 5s, 35 cycles in total; 72℃ extension for 5min. After the reaction, the PCR reaction solution was subjected to gel electrophoresis. Figure 5 It can be seen that a single and bright NtActin band was detected in both the wild-type tobacco and the transgenic peony PlDGAT2 gene tobacco, while as for the amplified Hyg band, a single, bright, and clear band was only detected in the transgenic peony PlDGAT2 gene tobacco, but not in the wild-type tobacco. At the same time, when the PlDGAT2 gene was amplified, a single, bright, and clear band was only detected in the transgenic peony PlDGAT2 gene tobacco.

[0043] qRT-PCR identification: The total RNA of wild-type tobacco and the three transgenic tobacco leaves obtained in Example 3 was extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa), and the total RNA was reverse transcribed into cDNA using the HiScript III RTSuperMix forqPCR (+gDNA wiper) (Vazyme) kit. The reaction system was: 1.0 μL RNA, 4.0 μL 4×gDNA wiper Mix, 11.0 μL RNase Free dH2O; the reaction conditions were: 42°C for 2 min. After the reaction was completed, 4.0 μL 5×HiScript III qRT SuperMix was added to the reaction solution of the first step; the reaction conditions were: 37°C for 15 min, 85°C for 5 s. The cDNA obtained by reverse transcription was detected by qRT-PCR using the SYBR qPCR SuperMix Plus (Novoprotein) kit. On this basis, the tobacco NtActin (AB158612) gene was used as an internal reference (SEQ ID NO.5, SEQ ID NO.6), and specific primers for the PlDGAT2 gene (Forward Primer: 5'-TACAAGTGGTGGAAGCC-3' (SEQ ID NO.7), Reverse Primer: 5'-CATTGGATGTCGAAAGG-3' (SEQ ID NO.8)) were designed for qRT-PCR detection. Reaction system: 2μL cDNA, 12.5μLSYBR qPCR SuperMixPlus, 1μL Forward Primer, 1μL Reverse Primer, 8.5μL ddH2O. Reaction procedure: 95℃ pre-denaturation for 3min; 95℃ denaturation for 5s, 55℃ annealing for 30s, 72℃ extension for 30s, a total of 40 cycles; 72℃ extension for 10min. After the reaction, 2 -△△ Ct method was used to analyze the relative expression level of genes. The results of qRT-PCR identification showed that PlDGAT2 had a significantly higher expression level in transgenic tobacco ( Figure 6 ).

[0044] Example 6 Identification of the ability of transgenic 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 seeds were harvested. After the tobacco seeds were dried in an oven at 65°C, fatty acid extraction was performed on the seeds, GC-MS analysis was performed, and qualitative and quantitative analysis of the relevant fatty acids was performed. Figure 7The results showed that the total fatty acid content in the seeds of the transgenic tobacco with overexpression of the PlDGAT2 gene increased by 20% compared with the wild tobacco control, indicating that overexpression of PlDGAT2 can increase the fatty acid content in tobacco seeds. In addition, the composition of the transgenic tobacco seeds with overexpression of the PlDGAT2 gene was analyzed, and the results were as follows 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. At the same time, 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. The linoleic acid (C18:2) content in OE-L1 and OE-L2 tobacco seeds decreased by 5.90% and 9.01%, respectively. The composition of other fatty acids did not change significantly ( Figure 8 ).

[0046] In summary, the overexpression of PlDGAT2 can promote the accumulation of palmitic acid (C16:0) and α-linolenic acid (C18:3), and transgenic tobacco 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, the present invention provides a full-length cDNA sequence of the peony PlDGAT2 gene and its application in regulating the fatty acid composition in plant seeds. By transforming the constructed PlDGAT2 gene overexpression vector into tobacco for expression, a new tobacco germplasm with a strong ability to control the fatty acid composition in plant seeds is created.

Claims

1. A peony PlDGAT2 gene, characterized in that The nucleotide sequence of the peony PlDGAT2 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the peony PlDGAT2 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The amplification primers for cloning the full-length cDNA sequence of the peony PlDGAT2 gene according to claim 1 are characterized in that: The sequences of the amplification primers are shown in SEQ ID NO.9 and SEQ ID NO.

10.

4. Use of an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the peony P1DGAT2 gene according to claim 1 in increasing the total fatty acid content and / or regulating the fatty acid composition in plant seeds.

5. The use according to claim 4, characterized in that: The regulating the fatty acid composition in plant seeds refers to increasing the content of palmitic acid and / or α-linolenic acid in the seeds.

6. The use according to claim 4, characterized in that: The plants include tobacco.

7. The use according to claim 4, characterized in that: The recombinant vector is obtained by introducing the ORF sequence of the peony PlDGAT2 gene into a plant binary expression vector.

8. The use according to claim 7, characterized in that: The primer sequences used to amplify the ORF sequence of the Paeonia lactiflora PlDGAT2 gene are shown in SEQ ID NO.3 and SEQ ID NO.

4.

9. A method for identifying high fatty acid plant seeds, characterized in that: The following steps are involved: 1) identifying whether the plant seeds contain the peony PlDGAT2 protein with an amino acid sequence as shown in SEQ ID NO.2; 2) Identifying whether the plant seeds contain the peony PlDGAT2 gene with a nucleotide sequence as shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that 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.

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