Application of OvDGAT1 and OvPLA genes from Oryza sativa L. in efficient accumulation of dihydroxy fatty acids

Abstract

The invention discloses the application of OvDGAT1 gene and OvPLA gene from scutellaria baicalensis in the efficient accumulation of dihydroxy fatty acids, and belongs to the field of genetic engineering technology. The invention discloses that OvDGAT1-1, OvDGAT1-2 and OvPLA genes from scutellaria baicalensis are key genes for the efficient accumulation of dihydroxy fatty acids, and through an Agrobacterium-mediated transformation system, seed-specific overexpression verification is performed on Arabidopsis thaliana as the background, thereby verifying the important role played by OvDGAT1-1, OvDGAT1-2 and OvPLA genes in the efficient accumulation of dihydroxy fatty acids, providing a certain theoretical basis for the efficient accumulation of dihydroxy fatty acids, and being conducive to the efficient production of dihydroxy fatty acids in other crops using synthetic biology in the future, making up for the defects of plant-based lubricants currently on the market, and generating sustainable development, biodegradable and environmentally friendly plant-based lubricants.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of OvDGAT1 gene and OvPLA gene from Oryctolagus serrata in the efficient accumulation of dihydroxy fatty acids. Background Art

[0002] As one of the alternative energy sources for petroleum, vegetable oil has very significant advantages. First, vegetable oil is renewable. Second, the production and processing of plant-based industrial oil has little impact on the environment. It is an environmentally friendly industrial oil and is conducive to the sustainable development of human society. In the past 20 years, people have been committed to the research on the use of vegetable oil to replace fossil fuels in production practice, but the developed plant-based industrial oil products have poor performance and problems of low volatility and polyunsaturation. Studies have found that industrial oils made from castor oil have better performance, such as high melting point, boiling point, reaction activity and other special properties. Castor oil has such excellent properties due to the presence of hydroxy fatty acids, so more and more researchers have begun to pay attention to the industrial use of hydroxy fatty acid plants. Armendáriz et al. made castor oil into bio-based diesel and found that its performance is better than that of ordinary vegetable oil and mineral diesel. Specifically, biodiesel made from castor oil does not produce atomization when burned, and the brake thermal efficiency is higher than that of mineral-based diesel and biodiesel made from ordinary vegetable oil. This provides strong support for the development of castor oil in the production of biodiesel. Hydroxy fatty acids are also made into plant-based lubricants for use in automobile engines due to their excellent lubricating properties. At the same time, hydroxy fatty acids are used in the production of cosmetics and surfactants due to their safety, non-toxicity and high reactivity. The esterified derivatives of hydroxy fatty acids, polyesters, can also be used to make degradable plastics, nylon, etc. In addition, hydroxy fatty acids can also be used in the biomedical field, such as the preparation of nanoparticles, the treatment of blepharitis and the stimulation of labor.

[0003] Currently, plant-based lubricants from castor are widely used in the industrial field as an alternative energy source. However, castor has certain climate requirements for the planting area and cannot be planted on a large scale. The limited output leads to its high price. In addition, castor oil contains ricin and highly allergenic proteins, which makes plant-based lubricants from castor have certain safety risks.

[0004] Studies have found that the seed oil of radix strychnifolia and its closely related species, radix strychnifolia, contains two special dihydroxy ultra-long chain fatty acids, C24:1-2OH and C24:2-2OH, and the content of these two fatty acids in radix strychnifolia reaches more than 40%. Studies on the physical and chemical properties of radix strychnifolia seed oil have found that its high-temperature lubrication performance is better than that of castor oil due to the presence of these two special fatty acids. More importantly, radix strychnifolia seed oil does not contain ricin and any allergenic proteins, making it an ideal raw material for plant-based lubricants. The OvFAE1-1, OvFAE1-2 and OvFAH genes have been identified as key genes for the synthesis of these two dihydroxy fatty acids. However, the content of dihydroxy fatty acids produced in transgenic Arabidopsis seeds into which the above three genes were transferred was much lower than that in radix strychnifolia seeds, indicating that the mechanism of efficient accumulation of dihydroxy fatty acids in radix strychnifolia remains to be elucidated. Summary of the invention

[0005] The purpose of the present invention is to provide the application of OvDGAT1 gene and OvPLA gene from Oryza sativa in the efficient accumulation of dihydroxy fatty acids, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a certain theoretical basis for the efficient accumulation of dihydroxy fatty acids, which is conducive to the efficient production of dihydroxy fatty acids in other crops using synthetic biology in the future, expanding its production range and increasing its yield, making up for the defects of plant-based lubricants currently on the market, and generating sustainable, biodegradable and environmentally friendly plant-based lubricants.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of the OvDGAT1 gene and the OvPLA gene from Oryza sativa L. in improving the efficient accumulation of dihydroxy fatty acids in plant seeds. The OvFAE1-1 gene, the OvFAE1-2 gene and the OvFAH gene are simultaneously overexpressed in plants together with the OvDGAT1 gene and / or the OvPLA gene, so that dihydroxy fatty acids can be efficiently accumulated in the plant seeds.

[0008] The OvDGAT1 gene is OvDGAT1-1 gene and OvDGAT1-2 gene, the nucleotide sequence of the OvDGAT1-1 gene is shown in SEQ ID No.1, and the nucleotide sequence of the OvDGAT1-2 gene is shown in SEQ ID No.2;

[0009] The nucleotide sequence of the OvPLA gene is shown in SEQ ID No.5;

[0010] The nucleotide sequence of the OvFAE1-1 gene is shown in SEQ ID No.35;

[0011] The nucleotide sequence of the OvFAE1-2 gene is shown in SEQ ID No.36;

[0012] The nucleotide sequence of the OvFAH gene is shown in SEQ ID No.37.

[0013] Furthermore, the plant includes Arabidopsis thaliana.

[0014] Furthermore, the dihydroxy fatty acid is a C24:1-2OH type and a C24:2-2OH type dihydroxy fatty acid.

[0015] The present invention also provides a method for improving the efficient accumulation of dihydroxy fatty acids in plant seeds, comprising constructing a multi-gene recombinant expression vector that simultaneously expresses OvFAE1-1 gene, OvFAE1-2 gene and OvFAH gene as well as OvDGAT1 gene and / or OvPLA gene, and introducing the multi-gene recombinant expression vector into the plant;

[0016] The OvDGAT1 gene is OvDGAT1-1 gene and OvDGAT1-2 gene, the nucleotide sequence of the OvDGAT1-1 gene is shown in SEQ ID No.1, and the nucleotide sequence of the OvDGAT1-2 gene is shown in SEQ ID No.2;

[0017] The nucleotide sequence of the OvPLA gene is shown in SEQ ID No.5;

[0018] The nucleotide sequence of the OvFAE1-1 gene is shown in SEQ ID No.35;

[0019] The nucleotide sequence of the OvFAE1-2 gene is shown in SEQ ID No.36;

[0020] The nucleotide sequence of the OvFAH gene is shown in SEQ ID No.37.

[0021] Furthermore, the dihydroxy fatty acid is a C24:1-2OH type and a C24:2-2OH type dihydroxy fatty acid.

[0022] Furthermore, the plant includes Arabidopsis thaliana.

[0023] Furthermore, the introduction is to introduce the multi-gene recombinant expression vector into the plant using Agrobacterium transformation.

[0024] Furthermore, the Agrobacterium transformation method is an Agrobacterium-mediated inflorescence infection method.

[0025] The present invention discloses the following technical effects:

[0026] The invention discloses that OvDGAT1-1, OvDGAT1-2 and OvPLA genes derived from Oryza sativa are key genes for efficient accumulation of dihydroxy fatty acids, and verify the seed-specific overexpression of OvDGAT1-1, OvDGAT1-2 and OvPLA genes in Columbia wild-type Arabidopsis thaliana (Columbia, Col-0) through an Agrobacterium-mediated transformation system, thereby verifying the important role played by OvDGAT1-1, OvDGAT1-2 and OvPLA genes in the efficient accumulation of dihydroxy fatty acids.

[0027] The present invention provides a certain theoretical basis for the efficient accumulation of dihydroxy fatty acids. Analyzing this pathway is of great significance for the discovery of this new industrial raw material. By studying the key genes involved in the efficient accumulation of dihydroxy fatty acids in Achyranthes bidentata, it is beneficial to use synthetic biology to efficiently produce dihydroxy fatty acids in other crops in the future, expand its production range and increase yield, make up for the defects of plant-based lubricants currently on the market, and generate sustainable, biodegradable and environmentally friendly plant-based lubricants. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 Schematic diagram of the overexpression vectors of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 in the embodiments of the present invention;

[0030] Figure 2 The agarose gel image of the full-length (including promoter and terminator) of OvDGAT1-1 and OvDGAT1-2 genes after PCR amplification in the embodiment of the present invention, M is DL 5000 DNA Marker; Lanes 1 and 2 are OvDGAT1-1 gene bands with a length of 2596 bp; Lanes 3 and 4 are OvDGAT1-2 gene bands with a length of 3037 bp;

[0031] Figure 3 This is an agarose gel image of positive clone detection of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 vectors. M is DL 2000 DNA Marker. Lanes 1-6 are OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 vectors; lanes 7-12 are OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT 1-2 vectors. The length of the bands is 1334 bp.

[0032] Figure 4 This is an agarose gel image of the target gene detection after the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 vectors in the embodiment of the present invention were transformed into the Columbia wild-type Arabidopsis recipient material, M is DL 2000DNA Marker, WT is the negative control, the plasmid is the positive control, and the target fragment size is 1024bp; A is the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 vector, and B is the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 vector;

[0033] Figure 5 Schematic diagram of the overexpression vectors of OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-2 in the embodiments of the present invention;

[0034] Figure 6 This is an agarose gel image of the full-length OvPLA gene (including the promoter and terminator) after PCR amplification in the embodiment of the present invention, M is DL 5000 DNA Marker, and the length of the amplified target fragment band is 2834 bp;

[0035] Figure 7 This is an agarose gel image of positive clone detection of OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFA H+OvPLA+OvDGAT1-2 vectors. M is DL 2000 DNA Marker. Lanes 1-6 are OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 vectors; lanes 7-12 are OvFAE1-1+Ov FAE12+OvFAH+OvPLA+OvDGAT1-2 vectors. The length of the bands is 1334 bp.

[0036] Figure 8This is an agarose gel image of the target gene detection after the OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-2 vectors in the embodiment of the present invention were transformed into the Columbia wild-type Arabidopsis recipient material, M is DL 2000DNA Marker, WT is the negative control, the plasmid is the positive control, and the target fragment size is 888bp; A is the OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 vector, and B is the OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-2 vector;

[0037] Fig. 9 It is a bar graph showing the fatty acid content and composition of mature seeds of the T3 generation pure line after the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 vector is overexpressed in the seeds of the Columbia wild-type Arabidopsis recipient material in the embodiment of the present invention, compared with the control OvFAE1-1+OvFAE1-2+OvFAH vector;

[0038] Fig.10 It is a bar graph showing the fatty acid content and composition of mature seeds of the T3 generation pure line after the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 vector is overexpressed in the seeds of the Columbia wild-type Arabidopsis recipient material in the embodiment of the present invention, compared with the control OvFAE1-1+OvFAE1-2+OvFAH vector;

[0039] Fig.11 It is a bar graph showing the fatty acid content and composition of mature seeds of the T3 generation pure line after the OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 vector is overexpressed in the seeds of the Columbia wild-type Arabidopsis recipient material in the embodiment of the present invention, compared with the control OvFAE1-1+OvFAE1-2+OvFAH vector;

[0040] Fig.12 It is a bar graph showing the fatty acid content and composition of mature seeds of the T3 generation pure line after the OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-2 vector overexpressed in the seeds of the Columbia wild-type Arabidopsis recipient material in the embodiment of the present invention compared with the control OvFAE1-1+OvFAE1-2+OvFAH vector;

[0041] Fig.13This is a Q-PCR statistical graph of the relative gene expression in seeds after the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1, OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2, OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1, and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 vectors were transformed into Columbia wild-type Arabidopsis recipient materials in the embodiments of the present invention, and WT is the wild-type control;

[0042] Fig.14 This is a diagram of the conservative domain analysis of the OvDGAT1-1 and OvDGAT1-2 genes in the embodiments of the present invention;

[0043] Fig.15 This is a diagram of the conserved domain analysis of the OvPLA gene in the examples of the present invention. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] In the present embodiment, seed-specific overexpression was verified in Arabidopsis thaliana, thereby verifying the important role played by OvDGAT1-1, OvDGAT1-2 and OvPLA genes in the efficient accumulation of dihydroxy fatty acids. The Arabidopsis thaliana was Columbia wild-type Arabidopsis thaliana (Columbia, Col-0).

[0046] The NapinRed3 plant expression vector used in the embodiment of the present invention is constructed based on pBin19, in which the original NOS promotor is replaced by Napin promotor and the fluorescent marker protein DsRed is inserted. The OleosinRed3 plant expression vector is constructed based on pBin19, in which the original NOS promotor is replaced by Oleosin promotor and the fluorescent marker protein DsRed is inserted.

[0047] The OvFAE1-1+OvFAE1-2+OvFAH vector is constructed by using pBin19 as the entry vector, inserting the OvFAE1-1 gene, OvFAE1-2 gene and OvFAH gene into the entry vector, and has been published in the document "Li Xiangjunet. Discontinuous fatty acid elongation yields hydroxylated seed oil with improved function, Nature plants, 2018-09-12".

[0048] The above vectors and Columbia wild-type Arabidopsis thaliana (Columbia, Col-0) are provided by Professor Zhang Chunyu's laboratory at Huazhong Agricultural University, and they are promised to be distributed to the public within 20 years from the date of application.

[0049] The sequences of the OvFAE1-1 gene, OvFAE1-2 gene, and OvFAH gene are as follows:

[0050] OvFAE1-1 gene sequence (SEQ ID No.35):

[0051]

[0052] OvFAE1-2 gene sequence (SEQ ID No.36):

[0053]

[0054] OvFAH gene sequence (SEQ ID No.37):

[0055]

[0056] Example 1 Construction of recombinant expression vectors of OvDGAT1-1 and OvDGAT1-2

[0057] The OvDGA1-1 and OvDGAT1-2 genes were cloned using the cDNA of the donor material Oryza sativa as a template, and the target genes were amplified by PCR. The primer sequences used for amplification were:

[0058] OvDGAT1-1 gene: forward primer (SEQ ID No. 7): 5'-ATGCCGACGGATAACGGTA-3'; reverse primer (SEQ ID No. 8): 5'-TCAGGACATCGATCCTTTGC-3';

[0059] OvDGAT1-2 gene: forward primer (SEQ ID No. 9): 5'-GTATCCTCGTCCAGTTTCCA-3'; reverse primer (SEQ ID No. 10): 5'-AGTCCTTGTGAGACGGGTTT-3';

[0060] use RNA from mature seeds of P. rapa was extracted using the Super total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd.) and reverse transcribed using HiScript IIQ RT SuperMix forqPCR reverse transcription reagent (Nanjing Novozymes Biotech Co., Ltd.) to obtain cDNA, and the target fragment was amplified using Phanta Max Super-FidelityDNAPolymerase (Nanjing Novozymes Biotech Co., Ltd.).

[0061] The amplified target gene sequence is as follows:

[0062] OvDGAT1-1 gene (SEQ ID No. 1):

[0063]

[0064] OvDGAT1-2 gene (SEQ ID No. 2):

[0065] GTATCCTCGTCCAGTTTCCATTTTCATCAGTCTTCCCAAAATTCACTTTTTCTTCTGCATGCTTTTCG ATTACTTAAACCTAACTCTCTTTTGAAGCTGTTTTGTTAACT AGAAACCCGTCTCACAAGGACT (Among them, the underlined ones are the flanking sequences corresponding to the upstream and downstream primers).

[0066] The amino acid sequence of the protein encoded by the target gene is as follows:

[0067] Amino acids of the protein encoded by the OvDGAT1-1 gene (SEQ ID No. 3):

[0068] MPTDNGSADLDRLRRRKSRSDSSNGLLSDSVSGNGTSPSDDTGAPADEDAQGTANLAGDTGGERGGVEAFRPSVPAHRRVRESSLSSDAIFQQSHAGLFNLCLVLLIAVNGRLSIENLMTY GLLITPDFWFTSTSSLRDWPLFMCCLSLPIFPLASFIVEQMVLQKHISETVVIILHIIITMSEVLYPVYVTLRCDSTFLSGNTLMLITCIVWLKLVSYAHTNYDMRALANSADDKANPEVSY YANLKSFAYFMVAPTLCYQPSYPRSPCIRKGWVARQFAKQVIFTGLIGCMTEQYINPIVRNSKHPLNGDLLYAIERMLKLSVPIVYVWLCWFYCFFHLWLNILAELLRFGDREFYKDWWNA KSVGEYWRMWNMPAHKWMARHVYFPCLRRKIPKGSAMIISFFVSAVFHELLIGVPCRVFRLWAFMGIMFQVPLVFITNYLQERFGSMVGNMFFWFVFCILGQPMCVILYYHDMMNSKGSMS.

[0069] Amino acids of the protein encoded by the OvDGAT1-2 gene (SEQ ID No. 4):

[0070] MPTDNGGADLDRLRRRKSKSDSSNGLLSDSVLGTDTSPSDDAGAPADKDAQGTANLAGDTGIRETGGGGGDGEAAYVRFACRPSVPAHRRVRESPLSSDAIFKQSHAGLFNLCLVLLIAANGRL IIENLMKYGWLITPDIWFTSTSLQDWPLYMCCLSLSIFPLASFTVEKMVLQKCISETAAIILHIIITTTEILYPVYVTLRCDSAFLPGFGMMVLTCTVWLKLVSYAHTNYDMRTLANSADKANP EVSYNVNLKSLAYFMVAPTLCYQPSYPRSPCIRKGWVARQFVKMVILVGCIGFITGQYINPILRNSKHPLKGDLLYLIERVLKLSIPKLYVWLCWFYCFFHVWLNILAELLRFGDREFYKDWWN ARNVGEYWRMWNVPVHKWMARHVYFPCLRRKIPKVFAMIIAFFVSAVFHELCISVPFRLFRPWVFFGIMYQVPLVFITNYIQKRFGSMVGNMIYWFMFCILWQPLFVILYYHDVMNHRKGSMS.

[0071] Using the plant expression vector NapinRed3 inserted with the fluorescent marker protein DsRed as the backbone, an expression vector driven by the seed-specific promoter Napin for the target gene was constructed. Subsequently, the fragment containing the Napin promoter, the target gene, and the terminator was amplified and connected to the OvFAE1-1+OvFAE1-2+OvFAH vector by homologous recombination to form the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1-2 vectors ( Figure 1 , Figure 2 and Figure 3 ), and finally transferred into GV3301 Agrobacterium competent cells, as detailed below:

[0072] The OvDGA1-1 and OvDGAT1-2 fragments were amplified using Napin-OvDGAT1-1 and Napin-OvDGAT1-2 vector plasmids as templates. The PCR reaction amplification primers consisted of three parts: homology arms, restriction sites, and target gene homology sequences, which facilitated the subsequent vector construction operations.

[0073] OvDGAT1 fragment: forward primer sequence (SEQ ID No. 11): 5'-taagaggagtccaccgggcccCGATGAGCTCTCTTCATCGG-3'; reverse primer sequence (SEQ ID No. 12): 5'-atacatcggcgcgccgggcccCAGAATTCCGCTTGCCGTTA-3';

[0074] 50μL reaction system: 2×Phanta Max Buffer 25μL, Forward Primer (10μM) 2μL, Reverse Primer (10μM) 2μL, dNTP Mix (10mM each) 1μL, DNA template 2μL, ddH2O 18μL.

[0075] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 3 min, 35 cycles; extension at 72°C for 5 min; 25°C for 5 min.

[0076] The amplified products were detected by 1.5% agarose gel electrophoresis. The expected band size of the OvDGAT1-1 fragment was 2596 bp, and the expected band size of the OvDGAT1-2 fragment was 3037 bp ( Figure 2 ).

[0077] The amplified target fragment was recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) to recover the PCR stock solution and measure the concentration of the target fragment after purification and recovery.

[0078] The triple gene overexpression vector of OvFAE1-1+OvFAE1-2+OvFAH was digested (Kana resistance).

[0079] Enzyme digestion system: buffer 6μL, plasmid DNA 50μL, FastAPFastDigestenzyme(s) 1μL, ApaI FastDigest enzyme(s) 1μL, ddH2O 2μL.

[0080] The reaction solution was centrifuged and mixed, and placed in a 37°C water bath for 30 min. After the reaction, DNA was recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) and the concentration was measured.

[0081] The target gene fragment and the enzyme-cut overexpression vector were connected using ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.).

[0082] Transform the ligated product into DH5α E. coli competent cells:

[0083] (1) Thaw cloning competent cells on ice;

[0084] (2) Take 10 μL of the recombinant product and add it to 100 μL of competent cells. Mix by gently tapping the tube wall (do not oscillate to mix). Incubate on ice for 30 min.

[0085] (3) After heat shock at 42°C in a water bath for 45 seconds, the mixture was immediately cooled on ice for 5 minutes;

[0086] (4) Add 700 μL LB medium (without antibiotics) and shake at 37°C for 1 h (speed 200 r / min);

[0087] (5) Preheat the Kana-resistant LB plate solid culture medium in a 37°C incubator;

[0088] (6) Centrifuge at 5000 rpm for 5 min, discard 600 μL of supernatant, resuspend the remaining cells, and spread them evenly on a plate containing Kana resistance using a sterile spreader;

[0089] (7) Incubate the cells in an incubator at 37°C for 12-16 hours.

[0090] For positive identification of monoclones, pick a single clone on the transformation plate and perform positive identification of bacterial solution PCR.

[0091] The forward primer sequence for PCR detection of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 vector was (SEQ ID No. 17): 5'-TCTACAGAAACATGGGTGCG-3'; the reverse primer sequence for PCR detection of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 vector was (SEQ ID No. 18): 5'-GACGCAGAACAGTTGAACAATG-3';

[0092] Bacterial liquid PCR 12μL reaction system: 2×Taq Master Mix (Dye Plus) 6μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, bacterial liquid 2μL, ddH2O 2μL.

[0093] The PCR reaction conditions of the bacterial solution were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min 30 s, 34 cycles; extension at 72°C for 5 min; and 25°C for 5 min.

[0094] The PCR reaction product was detected by 1% agarose gel electrophoresis, and the band at 1334 bp was a positive single clone ( Figure 3 ), and the transformed DH5α bacterial solution of the positive clones was directly sent to Wuhan Qingke Biotechnology Co., Ltd. for first-generation sequencing.

[0095] The clones whose sequencing results were completely consistent with the genome sequence were selected for expansion and culture, and the plasmid was extracted using TIANprep RapidMini Plasmid Kit (Tiangen Biochemical Technology Co., Ltd.). The plasmid was transformed into Agrobacterium GV3301, and the specific steps were as follows:

[0096] Take GV3301 Agrobacterium competent cells and melt them on ice. Add 0.01-1μg recombinant plasmid to 100μL competent cells. Place on ice for 5 minutes, transfer to liquid nitrogen and place for 5 minutes, 37℃ water bath for 5 minutes, ice for 5 minutes, add 400μL non-antibiotic LB, and incubate on a shaker at 28℃ for 1 hour. Take 100μL of transformation product and spread it on the resistant LB plate with Kana and Gent. Culture at 28℃ for 36-48h. Pick a single clone for detection. The primers and test steps used are consistent with the above positive identification of E. coli.

[0097] Example 2 Construction of recombinant expression vectors for simultaneous expression of OvDGAT1-1 and OvPLA and OvDGAT1-2 and OvPLA

[0098] The OvPLA gene was cloned using the cDNA of the donor material Oryza sativa as a template, and the target gene was amplified by PCR. The primer sequences used for amplification were:

[0099] OvPLA gene: forward primer (SEQ ID No. 13): 5'-CAATTTCTCACCAACCCTAA-3'; reverse primer (SEQ ID No. 14): 5'-TGACACTTACACTTGGCACA-3';

[0100] use RNA from mature seeds of P. rapa was extracted using the Super total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd.) and reverse transcribed using HiScript IIQ RT SuperMix forqPCR reverse transcription reagent (Nanjing Novozyme Biotechnology Co., Ltd.) to obtain cDNA, and the target fragment was amplified using Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotechnology Co., Ltd.).

[0101] The amplified target gene sequence is as follows:

[0102] OvPLA gene (SEQ ID No.5):

[0103] CAATTTCTCACCAACCCTAATTTCCCCAAATTGTGTACTACGGATTTCTTCATCTCACATTGGATTAA CAAAACGCTATCGATTCAACT AACGCGTCTTACCTTTGTAATGTGCCAAGTGTAAGTGTCA (Among them, the underlined ones are the flanking sequences corresponding to the upstream and downstream primers).

[0104] The amino acid sequence of the protein encoded by the OvPLA gene is shown in SEQ ID No.6:

[0105] .

[0106] The plant expression vector OleosinRed3 inserted with the fluorescent marker protein DsRed was used as the backbone to construct an expression vector driven by the seed-specific promoter Ole for the target gene. Subsequently, the fragment containing the Ole promoter, the target gene, and the terminator was amplified and connected to the OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 vector by homologous recombination to form OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-1 and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1-2 vectors ( Figure 5 , Figure 6 and Figure 7 ), and finally transferred into GV3301 Agrobacterium competent cells, as detailed below:

[0107] The OvPLA fragment was amplified using the Ole-OvPLA vector plasmid as a template. The PCR reaction amplification primer consists of three parts: homology arm, restriction site, and target gene homology sequence, which is convenient for subsequent vector construction operations.

[0108] OvPLA fragment: forward primer sequence (SEQ ID No. 15): 5'-ggagtccaccatggtagatctGGAACTATCA AGTCTGTGAC-3'; reverse primer sequence (SEQ ID No. 16): 5'-taccttctactagtcagatctCATTGGATTC TTGATGCTAC-3';

[0109] 50μL reaction system: 2×PhantaMax Buffer 25μL, Forward Primer (10μM) 2μL, Reverse Primer (10μM) 2μL, dNTP Mix (10mM each) 1μL, DNA template 2μL, ddH2O 18μL.

[0110] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 51°C for 15 s, extension at 72°C for 3 min, 35 cycles; extension at 72°C for 5 min; 25°C for 5 min.

[0111] The amplified product was detected by 1% agarose gel electrophoresis, and the expected size of the OvPLA fragment band was 2834 bp ( Figure 6 ).

[0112] The amplified target fragment was recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) to recover the PCR stock solution and measure the concentration of the target fragment after purification and recovery.

[0113] The four-gene overexpression vector of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 (Kana resistance) was digested with enzymes.

[0114] Enzyme digestion system: buffer 6μL, plasmid DNA 50μL, FastAPFastDigest enzyme(s) 1μL, ApaI FastDigest enzyme(s) 1μL, ddH2O 2μL.

[0115] The reaction solution was centrifuged and mixed, and placed in a 37°C water bath for 30 min. After the reaction, DNA was recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) and the concentration was measured.

[0116] The target gene fragment and the enzyme-cut overexpression vector were connected using ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.).

[0117] The ligated product was transformed into DH5α E. coli competent cells, following the same steps as in Example 1.

[0118] For positive identification of monoclones, pick a single clone on the transformation plate and perform positive identification of bacterial solution PCR.

[0119] The forward primer sequence for PCR detection of OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1 vector bacterial solution (SEQ ID No. 17) is: 5'-TCTACAGAAACATGGGTGCG-3'; the reverse primer sequence for bacterial solution detection (SEQ ID No. 18) is: 5'-GACGCAGAACAGTTGAACAATG-3';

[0120] Bacterial liquid PCR 12μl reaction system: 2×Taq Master Mix (Dye Plus) 6μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, bacterial liquid 2μL, ddH2O 2μL.

[0121] The PCR reaction conditions of the bacterial solution were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min 30 s, 34 cycles; extension at 72°C for 5 min; and 25°C for 5 min.

[0122] The PCR reaction product was detected by 1% agarose gel electrophoresis, and the band at 1334 bp was a positive single clone ( Figure 7 ), and the transformed DH5α bacterial solution of the positive clones was directly sent to Wuhan Qingke Biotechnology Co., Ltd. for first-generation sequencing.

[0123] The clones whose sequencing results were completely consistent with the genome sequence were selected for expansion and culture, and the plasmids were extracted using TIANprep RapidMini Plasmid Kit (Tiangen Biochemical Technology Co., Ltd.). The plasmids were transformed into Agrobacterium GV3301, and the specific steps were the same as those in Example 1.

[0124] Example 3 GV3301 Agrobacterium-mediated transformation of Arabidopsis

[0125] The transformed Agrobacterium was propagated and a single clone plaque was picked. 400 μL of LB containing Kana and Gent resistance was added and placed in a 28°C shaker at 200 r / min overnight. The 400 μL bacterial solution after small shaking was transferred to 500 mL of LB containing Kana and Gent antibiotics and placed in a 28°C shaker at 200 r / min overnight. The OD value was measured the next morning and LB was used as a blank control. 600 When the m / z is between 1.5 and 3.0, collect the cells by centrifugation at 4000 r / min for 15 min at room temperature, discard the supernatant, resuspend the cells with 500 mL of 5% freshly prepared sucrose solution, add SilwtL-77 to a final concentration of 0.05%, and mix immediately.

[0126] Plant treatment before transformation: Seedlings cut 7-10 days after the main flower axis is cut can be used for transformation experiments. At this time, the plant height is about 10-15cm, and the largest inflorescence has produced the first silique. Water thoroughly the day before transformation, cut off all the fruit pods and opened flowers of the pre-transformed seedlings, leaving only the flower buds, and seal the soil in the flowerpot with wide tape.

[0127] Transformation: Place the Arabidopsis plants prepared above upside down in the transformation solution, making sure that all flowers are immersed in the Agrobacterium culture solution and gently stir for about 30 seconds. Note that the time should not be too long, otherwise the plants will be damaged; the distance between the rosette leaves and the culture solution is 2 cm, and the same method is used to transform again after an interval of 7 days to improve the transformation rate.

[0128] Treatment of transformed plants: Take the plants out of the infiltration solution, cover them with a plastic bag and seal them, keep them away from light and keep them humid. After 24 hours, remove the plastic bag, stand the flower pot upright, and culture normally. When the siliques turn yellow, collect the seeds, dry them for 1 week, and store them in a refrigerator at 4°C.

[0129] The above method was used to stably genetically transform the constructed series of overexpression vectors into wild-type (WT) Arabidopsis. After harvesting T0 generation seeds, red fluorescence was used as a screening marker to screen T1 generation seeds and sow them. After germination, DNA of T1 generation transgenic Arabidopsis plants was extracted for genotype identification, with WT as a negative control and the plasmid of the overexpression vector transferred into each transgenic material as a positive control ( Figure 4 and Figure 8 ).

[0130] A certain amount of leaf samples of transgenic Arabidopsis lines were taken to extract DNA using the CTAB method;

[0131] The OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 vector was detected using positive detection primers (forward primer sequence: 5'-GTCTGAAACCGATGCCCTT-3', SEQ ID No. 19, reverse primer sequence: 5'-CCACATGGCCTTGAGTATTG-3', SEQ ID No. 20).

[0132] 20μL PCR amplification system, 2×Taq Master Mix (Dye Plus) 10μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, DNA template 2μL, ddH2O 6μL.

[0133] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 1 min, 34 cycles; extension at 72°C for 30 s; 25°C for 5 min, and finally detection by 1.5% agarose gel electrophoresis. The target band is at the position of 1024 bp ( Figure 4 ).

[0134] The OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1 vector was detected using positive detection primers (forward primer sequence: 5'-CAGTTTACAAAGCTTCAGGTGG-3', SEQ ID No. 21, reverse primer sequence: 5'-CGTGGTGTTTCTCCATTTCGA-3', SEQ ID No. 22).

[0135] 20μL PCR amplification system, 2×Taq Master Mix (Dye Plus) 10μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, DNA template 2μL, ddH2O 6μL.

[0136] PCR reaction conditions: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 56℃ for 15s, extension at 72℃ for 1min, 34 cycles; extension at 72℃ for 30s; 25℃ for 5min, and finally detection by 1.5% agarose gel electrophoresis. The target band is at 888bp ( Figure 8 ).

[0137] Example 4 Expression of OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDGAT1 after transformation into wild-type Arabidopsis

[0138] use Super total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd.) was used to extract total RNA from the siliques of T3 generation OvFAE1-1+OvFAE1-2+OvFAH+OvDGAT1 and OvFAE1-1+OvFAE1-2+OvFAH+OvPLA+OvDG AT1 transgenic Arabidopsis pure line materials 15 days after flowering, and the RNA concentration was measured.

[0139] According to the concentration of sample RNA, adjust the sample concentration with RNase-free ddH2O so that the total RNA injection volume is 12μL, add 4×gDNA Wiper Mix 4μL, mix gently with a pipette, and incubate at 42℃ for 2min.

[0140] Add 4 μL of 5×HiScript II qRT SuperMix II directly to the above reaction tube, mix gently by pipetting, and then perform reverse transcription reaction: 50°C for 15 min, 85°C for 5 s.

[0141] Quantitative Real-time PCR was used to analyze the expression of OvFAE1-1, OvFAE1-2, OvFAH, OvDGAT1-1, OvDGAT1-2, and OvPLA genes. Primer5 was used to design the quantitative primers for these genes. The sequences of the quantitative primers are as follows:

[0142] OvFAE1-1 gene forward quantitative primer (SEQ ID No. 23): 5'-CCTACAGCTGTATGCGAAGC-3'; reverse quantitative primer (SEQ ID No. 24): 5'-TTCTGAAGCAGTCCCTCGGTG-3';

[0143] OvFAE1-2 gene forward quantitative primer (SEQ ID No. 25): 5'-CAAAAAGCAAGTGTCTCCAAGG-3'; reverse quantitative primer (SEQ ID No. 26): 5'-CGGGACGTAGGTTTCATCTCCC-3';

[0144] OvFAH gene forward quantitative primer (SEQ ID No. 27): 5'-TTATCCAGCACACCCTTGGC-3'; reverse quantitative primer (SEQ ID No. 28): 5'-GAGGCGTTCACTGTCGTTAACG-3';

[0145] OvDGAT1-1 gene forward quantitative primer (SEQ ID No. 29): 5'-TGAGGGAGAGTTCACTCAGCT CT-3'; reverse quantitative primer (SEQ ID No. 30): 5'-AGGTGTGATCAACAACCCGTACG-3';

[0146] OvDGAT1-2 gene forward quantitative primer (SEQ ID No. 31): 5'-GGTACCGATACTTCCCCGTCAG-3'; reverse quantitative primer (SEQ ID No. 32): 5'-ACATAGGCAGCTTCTCCGTCC-3';

[0147] OvPLA gene forward quantitative primer (SEQ ID No. 33): 5'-GAAACACCACGAGAACGGAG-3'; reverse quantitative primer (SEQ ID No. 34): 5'-CCATCGTTTTGCGAATGGACTG-3';

[0148] qPCR reaction system: qPCR SYBR Green Master Mix 11.8μL, F / R Primer 0.6μL, template cDNA 2μL, ddH2O to 15μL.

[0149] qPCR reaction program: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 20 s, 45 cycles, melting curve stage default setting 1.

[0150] The expression of OvFAE1-1, OvFAE1-2, OvFAH, OvDGAT1-1, OvDGAT1-2, and OvPLA genes in transgenic plants. Compared with wild-type materials, these genes were overexpressed in transgenic materials ( Fig.13 ).

[0151] The OvDGAT1-1 gene is located on chromosome 4 of Atractylodes lancea, and its physical position is 19016314-19019091; after the OvDGAT1-1 gene is co-expressed with OvFAE1-1, OvFAE1-2, and OvFAH genes in wild-type Arabidopsis thaliana, the content of dihydroxy fatty acids in seeds can be significantly increased, indicating that the OvDGAT1-1 gene is a key gene for the efficient accumulation of dihydroxy fatty acids in Atractylodes lancea. The OvDGAT1-2 gene is located on chromosome 4 of A. rapa, and its physical location is 18826845-18828428. When the OvDGAT1-2 gene is co-expressed with the OvFAE1-1, OvFAE1-2, and OvFAH genes in wild-type Arabidopsis, the contents of two types of dihydroxy fatty acids, C24:1-2OH and C24:2-2OH, in seeds can be significantly increased, indicating that the OvDGAT1-2 gene is a key gene for the efficient accumulation of dihydroxy fatty acids in A. rapa. Fig. 9 , Fig.10 ). OvDGAT1-1 and OvDGAT1-2 have a typical conserved domain ( Fig.14 ), so this gene may have acyltransferase function. The OvPLA gene is on chromosome 11 of A. rapa, and its physical location is 97034222-97040644; the OvPLA gene can play a synergistic role with the OvDGAT1-1 or OvDGAT1-2 genes to further increase the content of dihydroxy fatty acids in transgenic Arabidopsis, indicating that the OvPLA gene is also a key gene for the efficient accumulation of dihydroxy fatty acids in A. rapa, and these genes have a higher substrate preference for 24-carbon dihydroxy fatty acids than other types of fatty acids ( Fig.11 and Fig.12 The OvPLA gene also has a typical structural domain ( Fig.15 ), so this gene may have the function of phospholipase.

[0152] The OvPLA gene encodes a phospholipase, which is located upstream of TAG synthesis. During the fatty acid chain acyl editing process, the PLA gene can promote the dissociation of fatty acid chains on PC into the Kennedy pathway to form TAG, thus helping to increase the content of hydroxy fatty acids in transgenic seeds. The OvDGAT1 gene encodes a diacylglycerol acyltransferase, which has a preference for hydroxy fatty acid substrates and can specifically acylate TAG containing hydroxy acyl chains for storage. These results indicate that the OvDGAT1 and OvPLA genes can be used as key genes to increase the content of dihydroxy fatty acids in transgenic seeds through genetic engineering.

[0153] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of OvDGAT1 gene and OvPLA gene from Oryctolagus serrata to improve the efficient accumulation of dihydroxy fatty acids in plant seeds, characterized in that: Overexpressing the OvFAE1-1 gene, OvFAE1-2 gene and OvFAH gene simultaneously with the OvDGAT1 gene, or overexpressing the OvFAE1-1 gene, OvFAE1-2 gene and OvFAH gene simultaneously with the OvDGAT1 gene and the OvPLA gene in plants can efficiently accumulate dihydroxy fatty acids in the seeds of the plants; The OvDGAT1 gene is the OvDGAT1-1 gene or the OvDGAT1-2 gene, the nucleotide sequence of the OvDGAT1-1 gene is shown in SEQ ID No.1, and the nucleotide sequence of the OvDGAT1-2 gene is shown in SEQ ID No.2; The nucleotide sequence of the OvPLA gene is shown in SEQ ID No.5; The nucleotide sequence of the OvFAE1-1 gene is shown in SEQ ID No.35; The nucleotide sequence of the OvFAE1-2 gene is shown in SEQ ID No.36; The nucleotide sequence of the OvFAH gene is shown in SEQ ID No.37; The dihydroxy fatty acids are C24:1-2OH type and C24:2-2OH type dihydroxy fatty acids; The plant is Arabidopsis thaliana.

2. A method for improving the efficient accumulation of dihydroxy fatty acids in plant seeds, characterized in that: The method comprises constructing a multi-gene recombinant expression vector that simultaneously expresses OvFAE1-1 gene, OvFAE1-2 gene, OvFAH gene and OvDGAT1 gene, or simultaneously expresses OvFAE1-1 gene, OvFAE1-2 gene, OvFAH gene, OvDGAT1 gene and OvPLA gene, and introducing the multi-gene recombinant expression vector into the plant; The OvDGAT1 gene is the OvDGAT1-1 gene or the OvDGAT1-2 gene, the nucleotide sequence of the OvDGAT1-1 gene is shown in SEQ ID No.1, and the nucleotide sequence of the OvDGAT1-2 gene is shown in SEQ ID No.2; The nucleotide sequence of the OvPLA gene is shown in SEQ ID No.5; The nucleotide sequence of the OvFAE1-1 gene is shown in SEQ ID No.35; The nucleotide sequence of the OvFAE1-2 gene is shown in SEQ ID No.36; The nucleotide sequence of the OvFAH gene is shown in SEQ ID No.37; The dihydroxy fatty acids are C24:1-2OH type and C24:2-2OH type dihydroxy fatty acids; The plant is Arabidopsis thaliana.

3. The method according to claim 2, characterized in that The introduction is to introduce the multi-gene recombinant expression vector into the plant by using Agrobacterium transformation method.

4. The method according to claim 3, characterized in that The Agrobacterium transformation method is an Agrobacterium-mediated inflorescence infection method.

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