CN107523584B - Transgenic method for improving oil content of plant nutritive tissue, expression vector and application - Google Patents

Abstract

The invention relates to a transgenic method for improving the oil content of plant nutritive tissues, an expression vector and application, belonging to the field of genetic engineering. SelectingLEC2、DGAT1、OLEO2AndGAT2four genes respectively participating in different oil synthesis and accumulation processes are connected with a proper promoter to construct a plurality of single/multiple gene expression vectors, and the constructed single/multiple gene expression vectors are introduced into plant nutritive tissues through agrobacterium transfection to enable the plant nutritive tissues to be ectopically or heterologously expressed, so that the oil content in the plant nutritive tissues is improved. In addition, the technology can also be used for improving fat-soluble substances related to synthesis and accumulation of oil and fat in plant nutritive tissuesAnd (4) production.

Description

Transgenic method for improving oil content of plant nutritive tissue, expression vector and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a transgenic method for improving the oil content of plant vegetative tissues, an expression vector and application.

Background

Vegetable oil has wide application, not only is the main source of edible oil, but also can be used for producing animal feed, soap, surfactant, cosmetics, paint, lubricating oil and biodiesel. However, due to the increase in population and the decrease in arable area, China faces a severe face of shortage of edible oil and chemical crude oil at present and for a long time in the future. It is highly desirable to make plants not only the major source of edible oils, but also potential plants for sustainable production of bioenergy and chemical feedstocks by improving and modifying the agronomic and quality traits of oil plants, including seed yield, oil content and their fatty acid components.

It is generally accepted that the production of oil in seeds involves three key elements: (1) efficiently converting the photosynthetic products to fatty acids; (2) efficient incorporation of fatty acids onto the glycerol backbone; (3) reducing degradation of fatty acids. In this connection, the main elements of the oil synthesizer are also present in the vegetative organs, because: (1) as the main constituent of oil, the main site of fatty acid synthesis is chloroplast or plastid, while plant leaves are rich in chloroplast and provide carbohydrates required for fatty acid synthesis; (2) the acyltransferase gene that assembles fatty acids onto a glycerol backbone to form oil is also expressed in vegetative organs, and this includes the diacylglycerol acyltransferase (DGAT1) gene that catalyzes the last step of the neutral lipid triacylglycerol synthesis; (3) under certain stress conditions or during plant senescence, the oil content in leaves or stems tends to increase significantly. In fact, vegetative organs such as leaves are a major component of harvestable biomass, and leaves provide the most significant carbon source for fatty acid synthesis, and thus may be ideal for oil accumulation if the elements of the oil synthesis machinery are functioning properly. However, leaves evolved during evolution to "source" tissues, to become highly specialized organs for carbohydrate synthesis and export, lacking the propensity for lipid accumulation. We assume that improper combination of components or inefficient operation of the oil synthesis machinery in the vegetative organs is one of the main causes of the low oil content trait, and if the three key genetic elements for controlling seed oil synthesis can be organically integrated into the vegetative organs, the oil synthesis and accumulation capacity will be enhanced.

In the classical Kennedy pathway, 3-phosphoglycerate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), and Acyl-CoA: diacylglycerol acyltransferase (DGAT), which is considered to be a key rate-limiting enzyme involved in the synthesis of seed oil and fat, assembles fatty acids at the sn-1, sn-2 and sn-3 positions of glycerol, respectively, and is found to be involved in the synthesis of TAG in senescent leaves. Some studies have been devoted to investigate the effect of the DGAT gene on oil synthesis in vegetative organs of plants under the control of constitutively expressed promoters (e.g. 35S), and the results show that overexpression of the AtDGAT1 gene in tobacco seedlings can increase the TAG content to 5.9 times that of the wild type, while in leaves, it can be up to 7 times that of the wild type.

It has been shown that overexpression of key enzyme genes or transcription factors in the fatty acid synthesis pathway in vegetative tissues can accelerate the conversion of photosynthetic products to fatty acids and the synthesis of lipids. Mendoza et al overexpressed the transcription factor LEAFY COTYLEDON2(LEC2) in Arabidopsis thaliana involved in the process of seed maturation and lipid accumulation regulation, and found that seed-specific mRNA was accumulated in vegetative tissues and the storage TAG content was significantly increased. In line with this, for expression of the 35S strong promoter-driven LEC2 gene, the oil content in the vegetative organs of Arabidopsis and tobacco can be effectively increased. But at the same time, somatic embryogenesis occurs in seedlings, and tissues are distorted and deformed, which affects the normal growth of transgenic plants. To solve this problem, Kim et al tried to over-express LEC2 gene in arabidopsis leaves by senescence-induced expression, and the result was that TAG content of transgenic plants was increased 3-fold compared to wild type and no obvious growth abnormality occurred. This suggests that organic binding of key genes to appropriate promoters is critical for the manipulation of lipid synthesis and accumulation in vegetative tissues of plants. However, the choice of which genome to incorporate and how to rationally construct a transcriptional unit to achieve gene expression based on vector tolerance and expression limitations remains a technical problem that needs to be considered and solved first.

The saccharomyces cerevisiae contains two genes of 3-phosphoglycerol acyltransferase (GPAT) GAT1 and GAT2 for encoding sn-1 position, catalyzes the first-step acylation reaction of glyceride biosynthesis pathway, although GAT1 and GAT2 belong to GPAT family genes and conserved acyltransferase characteristic sequences exist in polypeptides of yeast Gat1p and Gatp2, the genes GAT1 and GAT2 have obvious difference on a plurality of physiological effects of the yeast by constructing gene knockout or overexpression systems of GAT1 and GAT2 by taking the saccharomyces cerevisiae as a model organism. In addition, the Oleosin protein plays an important role in regulating the stability of the liposome in seeds, and the research of co-expression of the Oleosin gene and other genomes in the nutrition organs of oil crops is not carried out at present,

disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a transgenic method for increasing the oil content in vegetative tissues of plants, which can significantly increase the oil content in vegetative tissues of plants by ectopic or heterologous expression of a constructed expression vector in vegetative tissues of plants, and an expression vector and applications thereof.

An expression vector for increasing the oil content of vegetative tissue of plant contains the combination of GAT2, LEC2, DGAT1 and OLEO 2.

Further, the GAT2 gene is SCT1-YBL011w from Saccharomyces cerevisiae, or a gene with more than 90% of nucleotide sequence homology with SCT1-YBL011w, or a gene with more than 95% of amino acid sequence homology with SCT1-YBL011 w.

Further, the LEC2 gene is a transcription factor LEAFY COTYLEDON2 which is derived from arabidopsis thaliana and used for regulating lipid accumulation, or a gene with the nucleotide sequence homology of more than 90 percent with the transcription factor, or a gene with the amino acid sequence homology of more than 95 percent with the coding amino acid sequence of the transcription factor.

Furthermore, the DGAT1 gene is AT2G19450 derived from Arabidopsis thaliana, or a gene with more than 90% of nucleotide sequence homology with AT2G19450, or a gene with more than 95% of amino acid sequence homology with AT2G 19450.

Furthermore, the OLEO2 gene is AT5G40420 derived from Arabidopsis thaliana, or a gene with nucleotide sequence homology of more than 90% with AT5G40420, or a gene with amino acid sequence homology of more than 95% with AT5G 40420.

Furthermore, the promoter for starting LEC2 gene expression in the expression vector is 35S, and the terminator for terminating the gene expression is NOS; the promoter for starting the DGAT1 gene expression is 35S, and the terminator for terminating the gene expression is the 3' UTR of the At4g25710 gene; the promoter for starting the expression of the OLEO2 gene is the promoter of the At4g25700 gene, and the terminator for ending the expression of the gene is NOS; the promoter for promoting the expression of GAT2 gene was 35S, and the terminator for terminating the expression of the gene was NOS.

The invention also provides a transgenic method for improving the oil content of plant nutritive tissues by using the expression vector, which comprises the following steps:

step one, constructing the expression vector;

secondly, transforming the expression vector with agrobacterium tumefaciens, and obtaining positive agrobacterium tumefaciens after correct identification;

step three, taking the plant leaf explant for tissue culture, impregnating with positive agrobacterium tumefaciens, screening, continuing to culture until the plant leaf explant grows to roots, and transplanting for planting; or culturing the plant to the flowering phase, and performing dark culture after impregnating inflorescences with positive agrobacterium tumefaciens to finally obtain a transgenic positive plant;

and step four, taking leaves of the transgenic positive plants, performing molecular identification and lipid analysis, selecting the transgenic plants with high oil content, and collecting seeds after the plants are mature until transgenic plants with high oil content and stable inheritance are obtained.

The invention also protects the application of the expression vector in improving the oil content in plant nutritive tissues.

Further, the plant is tobacco, soybean, rape, sunflower, peanut, corn or alfalfa.

The invention also protects the application of the expression vector in improving the production of substances related to oil synthesis and accumulation in plant nutritive tissues, wherein the production of the substances related to oil synthesis and accumulation in the plant nutritive tissues is the production of fat-soluble substances.

The invention has the beneficial effects that:

1. the invention selects four genes GAT2, LEC2, DGAT1 and OLEO2 which are from arabidopsis thaliana or yeast and participate in different processes of fat synthesis and accumulation, connects with promoters with different strengths, leads the corresponding genes to be ectopically or heterologously expressed in plant nutritive tissues under the control of the promoters, effectively induces the vegetative cells to be transformed to embryonic cells, increases the content of oil in the vegetative organs by improving the activity of acyltransferase for synthesizing fat in the vegetative cells and reducing the activity of lipase for degrading the fat, and finally screens to obtain a gene combination which leads the plant nutritive tissues to have high oil content, thereby providing a molecular means for creating a system for producing the plant oil in the plant nutritive tissues.

2. The invention combines the genomes of the four key elements together to construct a plant expression vector which is transformed into plant tissues, so that the production capacity of oil in plant nutritive tissues is obviously improved. In 10 screened high-oil plants with tobacco as a research material, the highest TAG content can be increased by 21.59 times compared with the wild type. The condom system is expected to be applied to the production of plants such as soybean, rape, peanut and the like for increasing the oil content in the nutrition tissue or increasing part of fat-soluble substances.

Drawings

FIG. 1 is a map of the construction scheme of vector pBI121-35S MCS;

FIG. 2 is a map of a construction process of recombinant vector pYES 2-35S-NOS;

FIG. 3 is a map of the construction process of recombinant vector pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS;

FIG. 4 is a map of the construction process of recombinant vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO 2-NOS;

FIG. 5 is a map of the construction scheme of recombinant vector pYES2-DGAT1-OLEO 2;

FIG. 6 is a construction scheme of the recombinant vector pBI 12135S-GAT 2;

FIG. 7 is a construction scheme of the recombinant vector pBI121-LEC 2;

FIG. 8 is a flow chart of the construction of recombinant vector pBI121-DGAT1-OLEO2-GAT 2;

FIG. 9 is a construction scheme of the recombinant vector pBI121-LEC2-DGAT1-OLEO2-GAT 2;

FIG. 10 shows the restriction enzyme identification of the recombinant vector pBI121-LEC2-DGAT1-OLEO2-GAT 2; lane 1: StuI enzyme digestion; m: 1kb DNA ladder;

FIG. 11 is a diagram showing the PCR identification of the recombinant vector pBI121-LEC2-DGAT1-OLEO2-GAT 2; lane 1: the target fragment LEC2-DGAT 1; m: 1kb DNA ladder;

FIG. 12 is a schematic view of a tobacco regeneration process;

FIG. 13 is the triacylglycerol content in transgenic tobacco; WT: wild Arabidopsis thaliana; 6-3,6-9,6-10,6-11,6-12,6-18,6-19, 6-20,6-21,6-24: high oil plants of pBI121-LEC2-DGAT1-OLEO2-GAT2 vector;

FIG. 14 is a technical roadmap for the construction of plant binary vectors.

Detailed Description

The invention is further illustrated by the following specific examples.

Example (b):

1. construction of transcription Unit and screening of target Gene

To construct different plant transcription units, 35S and BCH1, which can be constitutively expressed in vegetative organs, were selected as promoters and NOS as terminators in this study. In addition, a target gene involved in oil and fat synthesis is also selected: (i) a gene GAT2 encoding 3-phosphoglycerol acyltransferase in yeast, (ii) a gene encoding diacylglycerol acyltransferase (DGAT1), the former and the latter participating in the first and third acylation reactions of lipid synthesis, respectively; (iii) oleosin 2(OLEO2) encodes an oil body chimeric protein that maintains the integrity and stability of oil droplets; (iv) the regulation factor LEC2 for converting vegetative cells into embryonic cells is ectopically expressed in leaf blades, and can obviously increase the synthesis and accumulation of leaf oil.

2. Cloning of the Gene of interest

2.1 cloning of Yeast GAT2

The GAT2 (gene sequence shown in SEQ ID NO: 1 and encoded amino acid sequence shown in SEQ ID NO: 5) was amplified using yeast genome as template with primers GAT2FP BamHI and GAT2RP XhoI, and a cleavage site BamHI was introduced at5 'end and a cleavage site XhoI at 3' end of GAT2, and a bright band of about 2200bp was observed by gel electrophoresis, and the objective gene GAT2(SCT1-YBL011w) -BamHI-XhoI was recovered.

2.2 cloning of Arabidopsis thaliana OLEO2, DGAT1 and LEC2

Extracting total RNA of arabidopsis thaliana, carrying out reverse transcription to form cDNA, carrying out PCR amplification by using the cDNA of arabidopsis thaliana as a template and using primers ZZF60 (OLEO2 FP) and ZZF61(OLEO2 RP), ZZF64(DGAT1 FP) and ZZF65(DGAT1 RP), ZZF68(LEC2 FP) and ZZF69(LEC2 RP) respectively to obtain OLEO2(872bp), DGAT1 (1591) and LEC2(1116 bp). Wherein, the gene sequence of OLEO2 is shown in SEQ ID NO: 3, and the coded amino acid sequence is shown as SEQ ID NO: 7 is shown in the specification; the gene sequence of DGAT1 is shown as SEQ ID NO: 2, and the coded amino acid sequence is shown as SEQ ID NO: 6 is shown in the specification; the gene sequence of LEC2 is shown in SEQ ID NO: 4, and the coded amino acid sequence is shown as SEQ ID NO: shown in fig. 8. Then, using the purified and recovered product as a template, PCR amplification was carried out using primers ZZF62(OLEO2 FP-SacI) and ZZF63(OLEO2 RP-SacI), ZZF66(DGAT1 FP-BamHI) and ZZF67(DGAT1 RP-BamHI), ZZF70(LEC2 FP-XbaI) and ZZF71(LEC2 RP-SacI), respectively, to add single cleavage sites to the 5 '-end and 3' -end, respectively, thereby finally obtaining the objective genes EO2(AT5G40420) -SacI-SacI, DGAT1(AT2G19450) -BamHI-BamHI and LEC2-XbaI-SacI with cleavage sites.

3. Cloning of promoter BCH1

The beta-carotene hydroxylase (BCH1) gene, 3 'UTR-BCH 1-5' UTR, was amplified to introduce a promoter and terminator. PCR amplification is carried out by using an arabidopsis genome as a template and using primers ZZF56(BCH1 FP) and ZZF57(BCH1 RP), then a 2504bp bright band is amplified by using primers ZZF58(BCH1 FP-BamHI) and ZZF59(BCH1 RP-SacI) through PCR again by using a purified and recovered product as a template, and the target gene BCHI-BamHI-SacI with the enzyme cutting sites is recovered.

4. Modification of vectors

4.1 transformation of binary vector pBI121-35S MCS

The 35S promoter was obtained by PCR amplification using plasmid pBI121-GUS as a template and primers ZZF47(35S promoter FP) and ZZF48(35S promoter RP). Then using 35S promoter as template, using 3 pairs of primers ZZF47(35S promoter FP) and ZZF49(35S promoter RP MCS1), ZZF47(35S promoter FP) and ZZF50(35S promoter RP MCS2), ZZF47(35S promoter FP) and ZZF51(35S promoter RP MCS3) to carry out PCR amplification on the purified and recovered products in sequence, observing a bright band of about 1000bp by gel electrophoresis, recovering, and finally obtaining the target gene 35S-MCS. Wherein the multiple cloning site comprises BamHI, XmaI, SmaI, XhoI and SacI. The desired gene 35S MCS was inserted into the vector pBI121-GUS through the restriction sites HindIII and SacI as shown in FIG. 1, followed by transformation, culture and plasmid extraction. Performing gel electrophoresis on the enzyme digestion products of the binary vector pBI121-35S MCS extracted by ClaI, EcoRI, HindIII and SacI through enzyme digestion respectively, wherein the enzyme digestion fragments are 1595bp, 11313bp, 915bp and 11993bp respectively, the verification is correct, and the new binary vector pBI121-35S MCS is obtained after sequencing verification.

4.2 transformation of intermediate vector pYES2-35S-NOS

35S-NOS was obtained by PCR amplification using pBI121-35S MCS as a template and primers ZZF52(35S FP) and ZZF53(NOS RP). Then, using purified and recovered 35S-NOS-T as a template, carrying out PCR amplification by using primers ZZF54(35S FP-KpnI-ClaI-StuI) and ZZF55(NOS-T-HindIII-ClaI-EcoRI), adding multiple enzyme cutting sites at the 5 'end and the 3' end, finally obtaining a bright band of about 1600bp, and recovering to obtain the target gene 35S-NOS. The target gene 35S-NOS is inserted into the vector pYES2 through the restriction enzyme sites KpnI and EcoRI, and as shown in FIG. 2, transformation, culture, plasmid extraction, restriction enzyme digestion and sequencing verification are carried out to verify the correctness, so that the intermediate vector pYES2-35S-NOS is obtained.

4.3 transformation of intermediate vector pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS

Using Arabidopsis genome DNA as a template, PCR amplification was performed using primers ZZF56(BCH1 FP) and ZZF57(BCH1 RP) to obtain 3 'UTR-BCH 1-5' UTR. Wherein, the 3 'UTR is a terminator of the At4g25710 gene, and the 5' UTR is a promoter of the At4g25700 gene. Then, PCR amplification was carried out using primers ZZF58(BCH1 FP-BamHI) and ZZF59(BCH1 RP-SacI) using the purified and recovered 3 'UTR-BCH 1-5' UTR as a template, and BamHI and SacI cleavage sites were added to the 5 'end and 3' end. The target gene 3 'UTR-BCH 1-5' UTR was inserted into the vector pYES2-35S MCS-NOS through the restriction enzyme sites BamHI and SacI, and the correct insertion was verified, thereby obtaining an intermediate vector pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS, as shown in FIG. 3.

Carrying out double enzyme digestion on pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS vector by using KpnI, EcoRI, BamHI and SacI respectively, carrying out gel electrophoresis on the enzyme digestion product, and carrying out accurate digestion on 3662bp, 5817bp, 2504bp and 6975bp respectively. And the sequence is correct after sequencing verification.

5. Construction of intermediate vectors

5.1 construction of the intermediate vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS

The target gene OLEO2-SacI-SacI is inserted into the vector pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS through the restriction enzyme cutting site SacI, and an intermediate vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS is obtained, as shown in FIG. 4.

It should be noted that: since the vector pYES2-35S-3 'UTR-BCH 1-5' UTR-NOS is obtained by SacI single enzyme digestion, in order to prevent vector self-ligation, the vector fragment needs to be dephosphorylated. Then purified and recovered by using a PCR product purification kit.

And (3) carrier verification: the vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS is digested by SacI, EcoRI and SacI respectively, the digested products are subjected to gel electrophoresis, and the digested fragments are 880bp, 9229bp, 630bp and 9479bp respectively. pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS is used as a template, ZZF56(BCH1 FP) and ZZF61(OLEO2 RP) are used as primers, and a 3105bp bright band is amplified by PCR to verify the directionality of the DNA. And the sequence is correct after sequencing verification.

5.2 construction of intermediate vector pYES2-DGAT1-OLEO2

The target gene DGAT1-BamHI-BamHI was inserted into vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS by digestion site BamHI, and finally intermediate vector pYES2-35S-3 'UTR-DGAT 1-BCH 1-5' UTR-OLEO2-NOS, i.e., pYES2-DGAT1-OLEO2, was obtained as shown in FIG. 5.

The vector pYES2-35S-3 'UTR-BCH 1-5' UTR-OLEO2-NOS was obtained by BamHI single digestion, and in order to prevent vector self-ligation, the vector fragment was dephosphorylated. Then purified and recovered by using a PCR product purification kit.

And (3) carrier verification: the vector pYES2-DGAT1-OLEO2 is cut by BamHI, and the cut products are subjected to gel electrophoresis, and the cut fragments are 1575bp and 10109 bp. pYES2-DGAT1-OLEO2 is used as a template, ZZF52(35S FP) and ZZF65(DGAT1 RP) are used as primers, and a 2408bp bright band is amplified by PCR to verify the directionality of the light band. And the sequence is correct after sequencing verification.

6. Construction of binary expression vectors

6.1 route of technology (as shown in FIG. 14)

6.2 construction of Single Gene expression vectors

6.2.1 construction of the Single Gene expression vector pBI 12135S-GAT 2

The target gene GAT2-BamHI-XhoI is inserted into binary vector pBI121-35S MCS through double enzyme cutting sites BamHI and XhoI to form single gene expression vector pBI 12135S-GAT 2, the construction map is shown in figure 6, and then the transformation of Escherichia coli sensing state DH5 alpha is carried out, and enzyme cutting verification and gene sequencing are carried out.

6.2.2 construction of the Single Gene expression vector pBI121-LEC2

Target genes LEC2-XbaI-SacI are inserted into binary vectors pBI121-35S MCS through double enzyme cutting sites XbaI and SacI to form single gene expression vectors pBI121-LEC2, a construction map is shown in figure 7, then escherichia coli competence DH5 alpha transformation is carried out, and enzyme cutting verification and gene sequencing are carried out.

And (3) carrier verification: the vector pBI 12135S-GAT 2 was digested with BamHI, XhoI and SacI, XbaI, respectively, and the digested fragments were subjected to gel electrophoresis to obtain 646bp, 13316bp, 1104bp and 12858bp fragments, respectively. And the sequence is verified to be correct by sequencing.

6.3 construction of three Gene expression vector pBI121-DGAT1-OLEO2-GAT2

The target fragment DGAT1-OLEO2 is cut from the vector pYES2-DGAT1-OLEO2 through the restriction enzyme cutting site ClaI, and then inserted into a single-gene expression vector pBI121-GAT2 to form a three-gene expression vector pBI121-DGAT1-OLEO2-GAT2, and the construction map is shown in FIG. 8. As the monogenic expression vector pBI121-GAT2 contains two same restriction enzyme cutting sites ClaI, the vector fragment is obtained by adopting an incomplete restriction enzyme cutting method.

And (3) carrier verification: the vector pBI121-DGAT1-OLEO2-GAT2 was digested with ClaI, and the digested fragments were subjected to gel electrophoresis, and the fragments were 3118bp, 5859bp, and 12053 bp. And the sequence is verified to be correct by sequencing; cutting pBI121-DGAT1-OLEO2-GAT2 with StuI and NotI enzyme, and performing gel electrophoresis on the cut enzyme products to obtain 4640bp and 16390bp cut fragments; a2295 bp bright band was PCR-amplified using pBI121-DGAT1-OLEO2-GAT2 as a template and GAT2FP BamHI and GAT2RP XhoI as primers. The carrier verification is correct.

6.4 construction of four Gene expression vectors

6.4.1 LEC2 Gene amplification

PCR amplification is carried out by taking a single gene expression vector pBI121-LEC2 as a template and using primers ZZF72(LEC2 PTU FP) and ZZF73(LEC2 PTU RP), then PCR amplification is carried out again by taking a purified and recovered product as a template and using primers ZZF74(LEC2 PTU FP-StuI) and ZZF75(LEC2 PTU RP-StuI) to obtain a 2249bp bright band, and recovery is carried out to obtain LEC2 PTU-StuI-StuI.

6.4.2 vector construction

Separately digesting LEC2 PTU-StuI-StuI and pBI121-DGAT1-OLEO2-GAT2 by StuI, and recovering digested products. Among them, for the vector fragment of not less than 20kb, the vector fragment is easily damaged mechanically during recovery, and the dragging phenomenon occurs, so it is necessary to extract the dephosphorylated vector fragment to remove the dephosphorylation reaction mixture. Then, the recovered LEC2 PTU-StuI-StuI and pBI121-DGAT1-OLEO2-GAT2 gene fragments are connected to finally obtain a four-gene expression vector pBI121-LEC2-DGAT1-OLEO2-GAT2, and the construction map is shown in FIG. 9.

6.4.3 vector validation

FIG. 10 shows that the vector pBI121-LEC2-DGAT1-OLEO2-GAT2 was digested with StuI, and the fragments digested were 2234bp and 21030bp, respectively, by gel electrophoresis. And the sequence is correct after sequencing verification. FIG. 11 shows that a 3808bp light band was PCR-amplified using pBI121-LEC2-DGAT1-OLEO2-GAT2 as template and ZZF68(LEC2 FP) and ZZF65(DGAT1 RP) as primers.

7. Transformation and identification of Agrobacterium tumefaciens GV3101

Preparing agrobacterium tumefaciens GV3101 competence, carrying out agrobacterium tumefaciens transformation on a plant expression vector pBI121-LEC2-DGAT1-OLEO2-GAT2, then extracting plasmids in the agrobacterium tumefaciens, and carrying out enzyme digestion or PCR verification on the plasmids.

8. Genetic transformation of tobacco

The leaf of field cultivated variety K326 is used as explant, plant expression vector pBI121-LEC2-DGAT1-OLEO2-GAT2 is introduced into tobacco through root nodule agrobacterium mediating process, and through 50 microgram/ml kanamycin (Kan) screening, transgenic tobacco plant is obtained.

The specific conversion steps are as follows: taking K326 young leaves growing in a greenhouse for about 2 months, carrying out surface disinfection, putting the leaves on 1/2MS culture medium, and pre-culturing for 2-3 days at 25 +/-1 ℃ in dark. Agrobacterium (after being infected with the transferred plant expression vector pBI121-LEC2-DGAT1-OLEO2-GAT2, the Agrobacterium is cultured for 2 days under the dark condition, then leaves are transferred to a screening culture medium, the culture is carried out for 40 days at 25 +/-1 ℃, 16h of light and 8h of dark, buds are cut and transferred to a rooting culture medium, and a transgenic tobacco plant is obtained after 14 days, as shown in figure 12.

9. Screening and identification of transgenic tobacco

Molecular characterization of 9.1T0 transgenic tobacco

The extracted genome DNA of the transgenic tobacco pBI121-LEC2-DGAT1-OLEO2-GAT2 is used as a template, and PCR verification is carried out by using primers ZZF47(35S promoter FP) and ZZF48(35S promoter RP), GAT2FP and GAT2RP, ZZF52(35S FP) and ZZF65(DGAT1 RP), ZZF56(BCH1 FP) and ZZF61(OLEO2 RP), ZZF68(LEC2 FP) and ZZF65(DGAT1 RP) to PCR amplify the insert 35S, GAT2, 35S-DGAT1, BCH 1-OLEO2 and LEC2-NOS, wherein the sizes of the insert are 911bp, 2295bp, 2431bp, 3125bp and 3808bp respectively. The result shows that 15 of 26 transgenic plants can simultaneously amplify 5 exogenous target gene segments, and the positive rate is 57.69%.

Lipid analysis of transgenic tobacco generations at 9.2T0

Extracting lipid in leaves of T0 generation transgenic tobacco plant pBI121-LEC2-DGAT1-OLEO2-GAT2, separating triacylglycerol by thin layer chromatography, and analyzing fatty acid components and relative content in triacylglycerol by gas chromatography.

As a result, among 15 positive transformed plants, 10 high oil plants 6-3,6-9,6-10,6-11,6-12,6-18,6-19, 6-20,6-21 and 6-24 were selected. Compared with the wild, the content of TAG is respectively increased by 9.92, 18.16, 21.13, 18.71, 7.86, 17.56, 21.59, 9.16, 20.01 and 17.07 times, as shown in figure 13.

The primers used in the above examples and their nucleotide sequences are shown in Table 1 below.

Table 1: primer and nucleotide sequence table

Therefore, under the action of a proper promoter, the gene combination of LEC2, DGAT1, OLEO2 and GAT2 regulates and controls key factors in the processes of oil synthesis, accumulation and degradation, and the genes are subjected to synergistic expression and action, so that the oil content in arabidopsis thaliana or tobacco nutritive tissues can be obviously improved, the method can be applied to plants such as soybean, rape, peanut or corn, and the production of biofuel is expected to be promoted; or applying it in forage crops such as alfalfa, sorghum, etc. to increase the nutrition of forage.

It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will be able to make many insubstantial modifications and adaptations to the invention without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Zhejiang agriculture and forestry university

<120> transgenic method for improving oil content of plant nutritive tissue, expression vector and application

<130> 1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 2280

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

atgcctgcac caaaactcac ggagaaattt gcctcttcca agagcacaca gaaaactacg 60

aattacagtt ccatcgaggc caaaagcgtc aagacgtcgg ctgatcaggc atacatctac 120

caagagccta gcgctaccaa gaagatactt tactccatcg ccacatggct gttgtacaac 180

atcttccact gcttctttag agaaatcaga ggccggggca gtttcaaggt accgcaacag 240

ggaccggtga tctttgttgc ggctccgcat gctaaccagt tcgtcgaccc tgtaatcctt 300

atgggcgagg tgaagaaatc tgtcaacaga cgtgtgtcct tcttgattgc ggagagctca 360

ttaaagcaac cccccatagg gtttttggct agtttcttca tggccatagg cgtggtaagg 420

ccgcaggata atttgaaacc ggcagaaggt actatccgcg tagatccaac agactacaag 480

agagttatcg gccacgacac gcatttcttg actgattgta tgccaaaggg tctcatcggg 540

ttacccaaat caatgggatt tggagaaatc cagtccatag aaagtgacac gagtttgacc 600

ctaagaaaag agttcaaaat ggccaaacca gagattaaaa ctgctttact caccggcact 660

acttataaat atgccgctaa agtcgaccaa tcttgcgttt accatagagt ttttgagcat 720

ttggcccata acaactgcat tgggatcttt cctgaaggtg ggtcccacga cagaacaaac 780

ttgttgcccc tgaaagcagg tgtggcgatt atggctcttg gttgcatgga taagcatcct 840

gacgtcaatg ttaagattgt tccctgcggt atgaattatt tccatccaca taagttcagg 900

tcgagagcgg ttgttgaatt cggtgacccc attgaaatac cgaaggaact agtcgccaag 960

taccacaacc cggaaacgaa cagagatgca gtgaaagaat tattagatac catatcgaag 1020

ggtttacaat ccgttaccgt tacatgttct gattatgaaa ctttgatggt ggttcaaacg 1080

ataagaagac tatatatgac acaatttagc accaagttac cgttgccctt gattgtggaa 1140

atgaacagaa gaatggtcaa aggttacgaa ttctatagaa acgatcctaa aatagcggac 1200

ttgaccaaag atataatggc atataatgcc gccttgagac actataatct tcctgatcac 1260

cttgtggagg aggcaaaggt aaatttcgca aaaaacctcg gacttgtttt ttttagatcc 1320

atcgggctct gcatcctctt ttcgttagcc atgccaggta tcattatgtt ctcacctgtc 1380

ttcatattag ccaagagaat ttctcaagaa aaggcccgta ccgctttgtc caagtctaca 1440

gttaaaataa aggctaacga tgtcattgcc acgtggaaaa tcttgattgg gatgggattt 1500

gcgcccttgc tttacatctt ttggtccgtt ttaatcactt attacctcag acataaacca 1560

tggaataaaa tatatgtttt ttccgggtct tacatctcgt gtgttatagt cacgtattcc 1620

gccttaatcg tgggtgatat tggtatggat ggtttcaaat ctttgagacc actggtttta 1680

tctcttacat ctccaaaggg cttgcaaaag ctacaaaagg atcgtagaaa tctggcagaa 1740

agaataatcg aagttgtaaa taactttgga agcgaattat tccccgattt cgatagtgcc 1800

gccctacgtg aagaattcga cgtcatcgat gaagaggaag aagatcgaaa aacctcagaa 1860

ttgaatcgca ggaaaatgct aagaaaacag aaaataaaaa gacaagaaaa agattcgtca 1920

tcacctatca tcagccaacg tgacaaccac gatgcctatg aacaccataa ccaagattcc 1980

gatggcgtct cattggtcaa tagtgacaat tccctctcta acattccatt attctcttct 2040

acttttcatc gtaagtcaga gtcttcctta gcttcgacat ccgttgcacc ttcttcttcc 2100

tccgaatttg aggtagaaaa cgaaatcttg gaggaaaaaa atggattagc aagtaaaatc 2160

gcacaggccg tcttaaacaa gagaattggt gaaaatactg ccagggaaga ggaagaggaa 2220

gaagaagagg aagaagaaga agaggaagaa gaagaagaag ggaaagaagg agatgcgtag 2280

<210> 2

<211> 1591

<212> DNA

<213> Arabidopsis thaliana

<400> 2

cttctggatc cttcgaaatg gcgattttgg attctgctgg cgttactacg gtgacggaga 60

acggtggcgg agagttcgtc gatcttgata ggcttcgtcg acggaaatcg agatcggatt 120

cttctaacgg acttcttctc tctggttccg ataataattc tccttcggat gatgttggag 180

ctcccgccga cgttagggat cggattgatt ccgttgttaa cgatgacgct cagggaacag 240

ccaatttggc cggagataat aacggtggtg gcgataataa cggtggtgga agaggcggcg 300

gagaaggaag aggaaacgcc gatgctacgt ttacgtatcg accgtcggtt ccagctcatc 360

ggagggcgag agagagtcca cttagctccg acgcaatctt caaacagagc catgccggat 420

tattcaacct ctgtgtagta gttcttattg ctgtaaacag tagactcatc atcgaaaatc 480

ttatgaagta tggttggttg atcagaacgg atttctggtt tagttcaaga tcgctgcgag 540

attggccgct tttcatgtgt tgtatatccc tttcgatctt tcctttggct gcctttacgg 600

ttgagaaatt ggtacttcag aaatacatat cagaacctgt tgtcatcttt cttcatatta 660

ttatcaccat gacagaggtt ttgtatccag tttacgtcac cctaaggtgt gattctgctt 720

ttttatcagg tgtcactttg atgctcctca cttgcattgt gtggctaaag ttggtttctt 780

atgctcatac tagctatgac ataagatccc tagccaatgc agctgataag gccaatcctg 840

aagtctccta ctacgttagc ttgaagagct tggcatattt catggtcgct cccacattgt 900

gttatcagcc aagttatcca cgttctgcat gtatacggaa gggttgggtg gctcgtcaat 960

ttgcaaaact ggtcatattc accggattca tgggatttat aatagaacaa tatataaatc 1020

ctattgtcag gaactcaaag catcctttga aaggcgatct tctatatgct attgaaagag 1080

tgttgaagct ttcagttcca aatttatatg tgtggctctg catgttctac tgcttcttcc 1140

acctttggtt aaacatattg gcagagcttc tctgcttcgg ggatcgtgaa ttctacaaag 1200

attggtggaa tgcaaaaagt gtgggagatt actggagaat gtggaatatg cctgttcata 1260

aatggatggt tcgacatata tacttcccgt gcttgcgcag caagatacca aagacactcg 1320

ccattatcat tgctttccta gtctctgcag tctttcatga gctatgcatc gcagttcctt 1380

gtcgtctctt caagctatgg gcttttcttg ggattatgtt tcaggtgcct ttggtcttca 1440

tcacaaacta tctacaggaa aggtttggct caacggtggg gaacatgatc ttctggttca 1500

tcttctgcat tttcggacaa ccgatgtgtg tgcttcttta ttaccacgac ctgatgaacc 1560

gaaaaggatc tatgtcatga ggatccacta a 1591

<210> 3

<211> 872

<212> DNA

<213> Arabidopsis thaliana

<400> 3

attacaaaga aaataggtaa aaacaatttc tcattagctt acaatggcgg atacacaccg 60

tgtcgaccgt actgatagac actttcaatt tcagtcgccc tatgaaggcg gccgaggtca 120

aggtcagtat gaaggtgacc gtggttacgg tggtggcggt tacaagagca tgatgcctga 180

aagtggccca tctagtaccc aagtattgtc cctgttgatt ggagtccctg tcgtcggttc 240

gctacttgcc ttggctggat tacttctagc tggttcggtg atcggcttaa tggttgcttt 300

accactattt ctcctcttca gcccggttat agtcccagcg gctctaacta tcgggcttgc 360

aatgacaggc tttttagcct cggggatgtt cggtctaacc gggcttagct caatctcatg 420

ggtcatgaac tatcttcgtg ggacaaggag aactgtgcct gagcaattgg agtatgctaa 480

gaggagaatg gctgatgcgg ttggctacgc aggacaaaag ggcaaagaaa tgggccagca 540

tgtgcagaac aaggcccaag atgttaaaca atatgatatt tctaagccac atgacactac 600

cactaagggt catgagactc aggggaggac gacggctgca tgatgagttt tcagtatgaa 660

cggtagatat gtgttttcac tattatgtcg ttttttctgc attttcaata tgatgttatg 720

tgtttttttt gtttggcttt ttgttgaacc gtgtatgtgt tttatgtttt tgtaagcatg 780

aaagatcgca agtgttgtgg taatatttga atgtaataat atgataagtt gataaatcat 840

gggaacattt aaattaggtg gacatgttta gc 872

<210> 4

<211> 2249

<212> DNA

<213> Arabidopsis thaliana

<400> 4

attgaaggcc tgcaggtccc cagattagcc ttttcaattt cagaaagaat gctaacccac 60

agatggttag agaggcttac gcagcaggtc tcatcaagac gatctacccg agcaataatc 120

tccaggaaat caaatacctt cccaagaagg ttaaagatgc agtcaaaaga ttcaggacta 180

actgcatcaa gaacacagag aaagatatat ttctcaagat cagaagtact attccagtat 240

ggacgattca aggcttgctt cacaaaccaa ggcaagtaat agagattgga gtctctaaaa 300

aggtagttcc cactgaatca aaggccatgg agtcaaagat tcaaatagag gacctaacag 360

aactcgccgt aaagactggc gaacagttca tacagagtct cttacgactc aatgacaaga 420

agaaaatctt cgtcaacatg gtggagcacg acacacttgt ctactccaaa aatatcaaag 480

atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta atatccggaa 540

acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata gtggaaaagg 600

aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt gaagatgcct 660

ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg gaaaaagaag 720

acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact gacgtaaggg 780

atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga agttcatttc 840

atttggagag aacacggggg actctagaaa atggataact tcttaccctt tccctcttct 900

aacgcaaact ctgtccaaga actctctatg gatcctaaca acaatcgctc gcacttcaca 960

acagtcccta cttatgatca tcatcaggct cagcctcatc acttcttgcc tccgttttca 1020

tacccggtgg agcagatggc ggcggtgatg aatcctcagc cggtttactt atcggagtgt 1080

tatcctcaga tcccggttac gcaaaccgga agtgaattcg gttctctggt tggtaatcct 1140

tgtttgtggc aagagagagg tggttttctt gatccgcgta tgacgaagat ggcaaggatc 1200

aacaggaaaa acgccatgat gagatcaaga aacaactcta gccctaattc tagtccaagt 1260

gagttggttg attcaaagag acagctgatg atgcttaact tgaaaaataa cgtgcagatc 1320

tccgacaaga aagatagcta ccaacagtcc acatttgata acaagaagct tagggttttg 1380

tgtgagaagg aattgaagaa cagcgatgtt gggtcactcg ggaggatagt tctaccaaag 1440

agagatgcag aagcaaatct tccgaagcta tctgataaag aaggaatcgt tgtacagatg 1500

agagatgttt tctctatgca gtcttggtct ttcaaataca agttttggtc caataacaag 1560

agcagaatgt atgtcctcga gaacacagga gaatttgtga agcaaaatgg agctgagata 1620

ggagactttt taacaatata cgaggacgaa agcaagaatc tctacttcgc catgaatgga 1680

aattcgggaa aacaaaatga aggaagagaa aatgagtcga gggaaaggaa ccactacgaa 1740

gaggcaatgc ttgattacat accaagagac gaagaggaag cttccattgc aatgctcatc 1800

ggaaatctaa acgatcacta tcccatccct aacgatctca tggacctcac cactgacctt 1860

cagcaccatc aagccacgtc ctcatcaatg ccacctgagg atcacgcgta cgtgggttca 1920

tccgatgatc aggtgagctt taacgacttt gagtggtggt gagagctcga atttccccga 1980

tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 2040

gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 2100

gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 2160

gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 2220

gttactagat cgggaattca ggcctaatg 2249

<210> 5

<211> 759

<212> PRT

<213> Saccharomyces cerevisiae

<400> 5

Met Pro Ala Pro Lys Leu Thr Glu Lys Phe Ala Ser Ser Lys Ser Thr

1 5 10 15

Gln Lys Thr Thr Asn Tyr Ser Ser Ile Glu Ala Lys Ser Val Lys Thr

20 25 30

Ser Ala Asp Gln Ala Tyr Ile Tyr Gln Glu Pro Ser Ala Thr Lys Lys

35 40 45

Ile Leu Tyr Ser Ile Ala Thr Trp Leu Leu Tyr Asn Ile Phe His Cys

50 55 60

Phe Phe Arg Glu Ile Arg Gly Arg Gly Ser Phe Lys Val Pro Gln Gln

65 70 75 80

Gly Pro Val Ile Phe Val Ala Ala Pro His Ala Asn Gln Phe Val Asp

85 90 95

Pro Val Ile Leu Met Gly Glu Val Lys Lys Ser Val Asn Arg Arg Val

100 105 110

Ser Phe Leu Ile Ala Glu Ser Ser Leu Lys Gln Pro Pro Ile Gly Phe

115 120 125

Leu Ala Ser Phe Phe Met Ala Ile Gly Val Val Arg Pro Gln Asp Asn

130 135 140

Leu Lys Pro Ala Glu Gly Thr Ile Arg Val Asp Pro Thr Asp Tyr Lys

145 150 155 160

Arg Val Ile Gly His Asp Thr His Phe Leu Thr Asp Cys Met Pro Lys

165 170 175

Gly Leu Ile Gly Leu Pro Lys Ser Met Gly Phe Gly Glu Ile Gln Ser

180 185 190

Ile Glu Ser Asp Thr Ser Leu Thr Leu Arg Lys Glu Phe Lys Met Ala

195 200 205

Lys Pro Glu Ile Lys Thr Ala Leu Leu Thr Gly Thr Thr Tyr Lys Tyr

210 215 220

Ala Ala Lys Val Asp Gln Ser Cys Val Tyr His Arg Val Phe Glu His

225 230 235 240

Leu Ala His Asn Asn Cys Ile Gly Ile Phe Pro Glu Gly Gly Ser His

245 250 255

Asp Arg Thr Asn Leu Leu Pro Leu Lys Ala Gly Val Ala Ile Met Ala

260 265 270

Leu Gly Cys Met Asp Lys His Pro Asp Val Asn Val Lys Ile Val Pro

275 280 285

Cys Gly Met Asn Tyr Phe His Pro His Lys Phe Arg Ser Arg Ala Val

290 295 300

Val Glu Phe Gly Asp Pro Ile Glu Ile Pro Lys Glu Leu Val Ala Lys

305 310 315 320

Tyr His Asn Pro Glu Thr Asn Arg Asp Ala Val Lys Glu Leu Leu Asp

325 330 335

Thr Ile Ser Lys Gly Leu Gln Ser Val Thr Val Thr Cys Ser Asp Tyr

340 345 350

Glu Thr Leu Met Val Val Gln Thr Ile Arg Arg Leu Tyr Met Thr Gln

355 360 365

Phe Ser Thr Lys Leu Pro Leu Pro Leu Ile Val Glu Met Asn Arg Arg

370 375 380

Met Val Lys Gly Tyr Glu Phe Tyr Arg Asn Asp Pro Lys Ile Ala Asp

385 390 395 400

Leu Thr Lys Asp Ile Met Ala Tyr Asn Ala Ala Leu Arg His Tyr Asn

405 410 415

Leu Pro Asp His Leu Val Glu Glu Ala Lys Val Asn Phe Ala Lys Asn

420 425 430

Leu Gly Leu Val Phe Phe Arg Ser Ile Gly Leu Cys Ile Leu Phe Ser

435 440 445

Leu Ala Met Pro Gly Ile Ile Met Phe Ser Pro Val Phe Ile Leu Ala

450 455 460

Lys Arg Ile Ser Gln Glu Lys Ala Arg Thr Ala Leu Ser Lys Ser Thr

465 470 475 480

Val Lys Ile Lys Ala Asn Asp Val Ile Ala Thr Trp Lys Ile Leu Ile

485 490 495

Gly Met Gly Phe Ala Pro Leu Leu Tyr Ile Phe Trp Ser Val Leu Ile

500 505 510

Thr Tyr Tyr Leu Arg His Lys Pro Trp Asn Lys Ile Tyr Val Phe Ser

515 520 525

Gly Ser Tyr Ile Ser Cys Val Ile Val Thr Tyr Ser Ala Leu Ile Val

530 535 540

Gly Asp Ile Gly Met Asp Gly Phe Lys Ser Leu Arg Pro Leu Val Leu

545 550 555 560

Ser Leu Thr Ser Pro Lys Gly Leu Gln Lys Leu Gln Lys Asp Arg Arg

565 570 575

Asn Leu Ala Glu Arg Ile Ile Glu Val Val Asn Asn Phe Gly Ser Glu

580 585 590

Leu Phe Pro Asp Phe Asp Ser Ala Ala Leu Arg Glu Glu Phe Asp Val

595 600 605

Ile Asp Glu Glu Glu Glu Asp Arg Lys Thr Ser Glu Leu Asn Arg Arg

610 615 620

Lys Met Leu Arg Lys Gln Lys Ile Lys Arg Gln Glu Lys Asp Ser Ser

625 630 635 640

Ser Pro Ile Ile Ser Gln Arg Asp Asn His Asp Ala Tyr Glu His His

645 650 655

Asn Gln Asp Ser Asp Gly Val Ser Leu Val Asn Ser Asp Asn Ser Leu

660 665 670

Ser Asn Ile Pro Leu Phe Ser Ser Thr Phe His Arg Lys Ser Glu Ser

675 680 685

Ser Leu Ala Ser Thr Ser Val Ala Pro Ser Ser Ser Ser Glu Phe Glu

690 695 700

Val Glu Asn Glu Ile Leu Glu Glu Lys Asn Gly Leu Ala Ser Lys Ile

705 710 715 720

Ala Gln Ala Val Leu Asn Lys Arg Ile Gly Glu Asn Thr Ala Arg Glu

725 730 735

Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu

740 745 750

Glu Gly Lys Glu Gly Asp Ala

755

<210> 6

<211> 520

<212> PRT

<213> Arabidopsis thaliana

<400> 6

Met Ala Ile Leu Asp Ser Ala Gly Val Thr Thr Val Thr Glu Asn Gly

1 5 10 15

Gly Gly Glu Phe Val Asp Leu Asp Arg Leu Arg Arg Arg Lys Ser Arg

20 25 30

Ser Asp Ser Ser Asn Gly Leu Leu Leu Ser Gly Ser Asp Asn Asn Ser

35 40 45

Pro Ser Asp Asp Val Gly Ala Pro Ala Asp Val Arg Asp Arg Ile Asp

50 55 60

Ser Val Val Asn Asp Asp Ala Gln Gly Thr Ala Asn Leu Ala Gly Asp

65 70 75 80

Asn Asn Gly Gly Gly Asp Asn Asn Gly Gly Gly Arg Gly Gly Gly Glu

85 90 95

Gly Arg Gly Asn Ala Asp Ala Thr Phe Thr Tyr Arg Pro Ser Val Pro

100 105 110

Ala His Arg Arg Ala Arg Glu Ser Pro Leu Ser Ser Asp Ala Ile Phe

115 120 125

Lys Gln Ser His Ala Gly Leu Phe Asn Leu Cys Val Val Val Leu Ile

130 135 140

Ala Val Asn Ser Arg Leu Ile Ile Glu Asn Leu Met Lys Tyr Gly Trp

145 150 155 160

Leu Ile Arg Thr Asp Phe Trp Phe Ser Ser Arg Ser Leu Arg Asp Trp

165 170 175

Pro Leu Phe Met Cys Cys Ile Ser Leu Ser Ile Phe Pro Leu Ala Ala

180 185 190

Phe Thr Val Glu Lys Leu Val Leu Gln Lys Tyr Ile Ser Glu Pro Val

195 200 205

Val Ile Phe Leu His Ile Ile Ile Thr Met Thr Glu Val Leu Tyr Pro

210 215 220

Val Tyr Val Thr Leu Arg Cys Asp Ser Ala Phe Leu Ser Gly Val Thr

225 230 235 240

Leu Met Leu Leu Thr Cys Ile Val Trp Leu Lys Leu Val Ser Tyr Ala

245 250 255

His Thr Ser Tyr Asp Ile Arg Ser Leu Ala Asn Ala Ala Asp Lys Ala

260 265 270

Asn Pro Glu Val Ser Tyr Tyr Val Ser Leu Lys Ser Leu Ala Tyr Phe

275 280 285

Met Val Ala Pro Thr Leu Cys Tyr Gln Pro Ser Tyr Pro Arg Ser Ala

290 295 300

Cys Ile Arg Lys Gly Trp Val Ala Arg Gln Phe Ala Lys Leu Val Ile

305 310 315 320

Phe Thr Gly Phe Met Gly Phe Ile Ile Glu Gln Tyr Ile Asn Pro Ile

325 330 335

Val Arg Asn Ser Lys His Pro Leu Lys Gly Asp Leu Leu Tyr Ala Ile

340 345 350

Glu Arg Val Leu Lys Leu Ser Val Pro Asn Leu Tyr Val Trp Leu Cys

355 360 365

Met Phe Tyr Cys Phe Phe His Leu Trp Leu Asn Ile Leu Ala Glu Leu

370 375 380

Leu Cys Phe Gly Asp Arg Glu Phe Tyr Lys Asp Trp Trp Asn Ala Lys

385 390 395 400

Ser Val Gly Asp Tyr Trp Arg Met Trp Asn Met Pro Val His Lys Trp

405 410 415

Met Val Arg His Ile Tyr Phe Pro Cys Leu Arg Ser Lys Ile Pro Lys

420 425 430

Thr Leu Ala Ile Ile Ile Ala Phe Leu Val Ser Ala Val Phe His Glu

435 440 445

Leu Cys Ile Ala Val Pro Cys Arg Leu Phe Lys Leu Trp Ala Phe Leu

450 455 460

Gly Ile Met Phe Gln Val Pro Leu Val Phe Ile Thr Asn Tyr Leu Gln

465 470 475 480

Glu Arg Phe Gly Ser Thr Val Gly Asn Met Ile Phe Trp Phe Ile Phe

485 490 495

Cys Ile Phe Gly Gln Pro Met Cys Val Leu Leu Tyr Tyr His Asp Leu

500 505 510

Met Asn Arg Lys Gly Ser Met Ser

515 520

<210> 7

<211> 199

<212> PRT

<213> Arabidopsis thaliana

<400> 7

Met Ala Asp Thr His Arg Val Asp Arg Thr Asp Arg His Phe Gln Phe

1 5 10 15

Gln Ser Pro Tyr Glu Gly Gly Arg Gly Gln Gly Gln Tyr Glu Gly Asp

20 25 30

Arg Gly Tyr Gly Gly Gly Gly Tyr Lys Ser Met Met Pro Glu Ser Gly

35 40 45

Pro Ser Ser Thr Gln Val Leu Ser Leu Leu Ile Gly Val Pro Val Val

50 55 60

Gly Ser Leu Leu Ala Leu Ala Gly Leu Leu Leu Ala Gly Ser Val Ile

65 70 75 80

Gly Leu Met Val Ala Leu Pro Leu Phe Leu Leu Phe Ser Pro Val Ile

85 90 95

Val Pro Ala Ala Leu Thr Ile Gly Leu Ala Met Thr Gly Phe Leu Ala

100 105 110

Ser Gly Met Phe Gly Leu Thr Gly Leu Ser Ser Ile Ser Trp Val Met

115 120 125

Asn Tyr Leu Arg Gly Thr Arg Arg Thr Val Pro Glu Gln Leu Glu Tyr

130 135 140

Ala Lys Arg Arg Met Ala Asp Ala Val Gly Tyr Ala Gly Gln Lys Gly

145 150 155 160

Lys Glu Met Gly Gln His Val Gln Asn Lys Ala Gln Asp Val Lys Gln

165 170 175

Tyr Asp Ile Ser Lys Pro His Asp Thr Thr Thr Lys Gly His Glu Thr

180 185 190

Gln Gly Arg Thr Thr Ala Ala

195

<210> 8

<211> 362

<212> PRT

<213> Arabidopsis thaliana

<400> 8

Met Asp Asn Phe Leu Pro Phe Pro Ser Ser Asn Ala Asn Ser Val Gln

1 5 10 15

Glu Leu Ser Met Asp Pro Asn Asn Asn Arg Ser His Phe Thr Thr Val

20 25 30

Pro Thr Tyr Asp His His Gln Ala Gln Pro His His Phe Leu Pro Pro

35 40 45

Phe Ser Tyr Pro Val Glu Gln Met Ala Ala Val Met Asn Pro Gln Pro

50 55 60

Val Tyr Leu Ser Glu Cys Tyr Pro Gln Ile Pro Val Thr Gln Thr Gly

65 70 75 80

Ser Glu Phe Gly Ser Leu Val Gly Asn Pro Cys Leu Trp Gln Glu Arg

85 90 95

Gly Gly Phe Leu Asp Pro Arg Met Thr Lys Met Ala Arg Ile Asn Arg

100 105 110

Lys Asn Ala Met Met Arg Ser Arg Asn Asn Ser Ser Pro Asn Ser Ser

115 120 125

Pro Ser Glu Leu Val Asp Ser Lys Arg Gln Leu Met Met Leu Asn Leu

130 135 140

Lys Asn Asn Val Gln Ile Ser Asp Lys Lys Asp Ser Tyr Gln Gln Ser

145 150 155 160

Thr Phe Asp Asn Lys Lys Leu Arg Val Leu Cys Glu Lys Glu Leu Lys

165 170 175

Asn Ser Asp Val Gly Ser Leu Gly Arg Ile Val Leu Pro Lys Arg Asp

180 185 190

Ala Glu Ala Asn Leu Pro Lys Leu Ser Asp Lys Glu Gly Ile Val Val

195 200 205

Gln Met Arg Asp Val Phe Ser Met Gln Ser Trp Ser Phe Lys Tyr Lys

210 215 220

Phe Trp Ser Asn Asn Lys Ser Arg Met Tyr Val Leu Glu Asn Thr Gly

225 230 235 240

Glu Phe Val Lys Gln Asn Gly Ala Glu Ile Gly Asp Phe Leu Thr Ile

245 250 255

Tyr Glu Asp Glu Ser Lys Asn Leu Tyr Phe Ala Met Asn Gly Asn Ser

260 265 270

Gly Lys Gln Asn Glu Gly Arg Glu Asn Glu Ser Arg Glu Arg Asn His

275 280 285

Tyr Glu Glu Ala Met Leu Asp Tyr Ile Pro Arg Asp Glu Glu Glu Ala

290 295 300

Ser Ile Ala Met Leu Ile Gly Asn Leu Asn Asp His Tyr Pro Ile Pro

305 310 315 320

Asn Asp Leu Met Asp Leu Thr Thr Asp Leu Gln His His Gln Ala Thr

325 330 335

Ser Ser Met Thr Pro Glu Asp His Ala Tyr Val Gly Ser Ser Asp Asp

340 345 350

Gln Val Ser Phe Asn Asp Phe Glu Trp Trp

355 360

Claims (9)

1. An expression vector for improving the oil content of plant nutritive tissues, which is characterized in that: comprises a main component consisting ofGAT2、LEC2、DGAT1AndOLEO2the gene combination is composed of four genes.

2. The expression vector of claim 1, wherein the expression vector is selected from the group consisting of: the above-mentionedGAT2The gene is derived from Saccharomyces cerevisiaeSCT1-YBL011w。

3. The expression vector of claim 1, wherein the expression vector is selected from the group consisting of: the above-mentionedLEC2The gene is a transcription factor which is derived from arabidopsis and used for regulating and controlling the accumulation of greaseLEAFY COTYLEDON2。

4. The expression vector of claim 1, wherein the expression vector is selected from the group consisting of: the above-mentionedDGAT1The gene is derived from Arabidopsis thalianaAT2G19450。

5. The expression vector of claim 1, wherein the expression vector is selected from the group consisting of: the above-mentionedOLEO2The gene is derived from Arabidopsis thalianaAT5G40420。

6. An expression vector according to any one of claims 1 to 5 for increasing the oil content of vegetative tissue of a plant, wherein: in the expression vectorLEC2The promoter of gene expression is35SThe terminator for terminating the expression of the gene isNOS(ii) a Starting upDGAT1The promoter of gene expression is35SThe terminator for terminating the expression of the gene isAt4g25710Of genes3’UTR(ii) a Starting upOLEO2The promoter of gene expression isAt4g25700Promoters of genesThe terminator for terminating the expression of the gene isNOS(ii) a Starting upGAT2The promoter of gene expression is35SThe terminator for terminating the expression of the gene isNOS。

7. Use of the expression vector of claim 6 for increasing the oil content in vegetative tissue of a plant.

8. The use of claim 7, wherein: the plant is tobacco, soybean, rape, sunflower, peanut, corn or alfalfa.

9. Use of the expression vector of claim 6 for increasing production of a substance associated with oil synthesis and accumulation in vegetative tissue of a plant, wherein: the production of the substances related to the synthesis and accumulation of the plant nutrient tissue oil is the production of fat-soluble substances.

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