CN113502295B

CN113502295B - Application of TmLPCAT gene in improving content of triacylglycerol sn-2-position ultra-long chain fatty acid

Info

Publication number: CN113502295B
Application number: CN202110660152.4A
Authority: CN
Inventors: 张猛; 马世杰; 宋欢
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-06-09
Filing date: 2021-06-15
Publication date: 2022-06-07
Anticipated expiration: 2041-06-15
Also published as: CN113502295A

Abstract

The invention relates to an application of TmLPCAT gene in improving the content of triacylglycerol sn-2 bit ultra-long chain fatty acid. The TmLPCAT gene involved comprises a sequence selected from the group consisting of: a sequence shown as SEQ ID NO. 1; the gene with over 90 percent homology with SEQ ID NO.1 can be used for improving the content of the super-long chain fatty acid on the sn-2 position in the triacylglycerol in crops. The invention plays an important role in the accumulation of the plant erucic acid.

Description

Application of TmLPCAT gene in improving content of triacylglycerol sn-2 bit ultra-long chain fatty acid

Technical Field

The invention belongs to the field of gene function and crop gene engineering, and particularly relates to a function of a gene and a gene-coded enzyme in synthesis of triacylglycerol sn-2 fatty acid and application thereof.

Background

Erucic acid, chemical name is cis-13-docosaenoic acid (13-Docosenoic acid, (13Z) -), is an ultra-long chain monounsaturated fatty acid, and is mainly present in seed storage oil of Cruciferae (Cruciferae) and globeflower (Tropaeolaceae). Erucic acid is showing more and more important function in many fields of modern industry because of its characteristics of regeneration, wide use, high added value, large market demand and the like. At present, as a main production country of erucic acid, the annual output of the erucic acid in China can reach about one third of the total output in the world. Erucic acid production currently relies primarily on extraction from the seed oil of cruciferous oil crops, which have an erucic acid content of 30-50% more in their seed oil. Therefore, the increasing of the percentage of the erucic acid in the cruciferous oil crops has a very important meaning for reducing the production cost of the erucic acid and increasing the income of growers and related processing enterprises.

90% of the seed oil is triacylglycerol, and the glycerol skeleton has three positions of sn-1, sn-2 and sn-3 in sequence, and fatty acid is connected to form triacylglycerol (figure 1). However, in cruciferous oil crops including rape, erucic acid can be accumulated at the sn-1 and sn-3 positions of triacylglycerols of seeds, but very little ultra-long chain fatty acids including erucic acid can be bound at the sn-2 position. This was also demonstrated by the inventors' examination of the triacylglycerols of brassica napus seeds (fig. 2). This becomes the bottleneck that the content of erucic acid can not be improved by the traditional breeding means. A gene cloned from plumeria poacherae (Limnanthes douglasii) into lysophosphatidic acid acyltransferase (LPAAT), which can be used to increase the synthesis of erucic acid at the triacylglycerol sn-2 position of rape seeds, has been investigated (Lassner et al 1995). Lysophosphatidylcholine acyltransferase (LPCAT) can transfer acyl to sn-2 position of lysophosphatidylcholine to form phosphatidylcholine, and then affect acyl type and content of sn-2 of triacylglycerol via diacylglycerol. In some plants, LPCAT plays a very important role in the accumulation of triacylglycerols. The newly synthesized acyl-CoA, for the most part, forms phosphatidylcholine via LPCAT, rather than completing triacylglycerol assembly via the Kennedy pathway. Most of newly formed phosphatidylcholine in storage tissues is finally converted into triacylglycerol through diacylglycerol, but no report or invention that LPCAT can improve erucic acid at sn-2 position is found so far.

Tropaeolum majus (Tropaeolum majus) is a rare ornamental plant of the family of globeflower, and is native to Chilean. Erucic acid accounts for a very high proportion of triacylglycerols in which seeds are stored. Unlike rape and the like, which cannot assemble erucic acid efficiently to the sn-2 position of glycerol, the erucic acid content of the sn-1, sn-2 and sn-3 positions in triacylglycerol stored in tropaeolum seeds is very high, and the triacylglycerol can be formed. The inventors also demonstrated that the erucic acid content at the sn-2 position of the triacylglycerols of the tropaeolum seed can exceed 80% (FIG. 2). However, previous studies have demonstrated that LPAAT enzyme does not have erucic acid substrate preference in developing seeds of tropaeolum majus.

Disclosure of Invention

The invention aims to disclose the TmLPCAT gene (the Tropaeolum majus LPCAT gene) in the Tropaeolum majus seeds and the function of the coded protein thereof in the erucic acid accumulation process, and to improve the content of the erucic acid on the sn-2 position in the triacylglycerol of the seeds by applying the TmLPCAT gene to oil crops.

Therefore, the invention provides the application of the tropaeolum LPCAT gene for improving the content of the super-long-chain fatty acid at the sn-2 position in triacylglycerol in plants, wherein the tropaeolum LPCAT gene is selected from a sequence of one of the following: a sequence shown as SEQ ID NO. 1; a gene having homology of 90% or more with SEQ ID NO. 1.

The invention also provides an expression vector containing the Tropaeolum majus LPCAT gene.

Optionally, the expression vector is Pha-TmLPCAT-DsRed-pK7WG2D, wherein Pha is a Phaseolin promoter, TmLPCAT is the tropaeolum LPCAT gene of claim 1, DsRed is a fluorescent protein marker gene expression cassette, and pK7WG2D is an expression vector framework.

The invention also provides a bacterial cell containing the Tropaeolum majus LPCAT gene.

Optionally, the provided bacterial cells are escherichia coli and agrobacterium cells containing the eclipta alba LPCAT gene.

Further, the application of the enzyme coded by the Tropaeolum majus LPCAT gene for improving the content of the super-long chain fatty acid at the sn-2 position in the triacylglycerol in the crops is realized, and the enzyme coded by the Tropaeolum majus LPCAT gene is selected from one of the following sequences: a sequence shown as SEQ ID NO. 2; a sequence having more than 90% homology with SEQ ID NO. 2.

The invention discloses a function of the gene in contributing to the accumulation of sn-2 erucic acid in triacylglycerol in seeds of a model plant Arabidopsis thaliana high erucic acid type strain. Due to the same genus of cruciferae, the lipid metabolism pathway of arabidopsis thaliana is very similar to that of the cabbage type rape which is the main oil crop at present, and meanwhile, the high erucic acid strain can better reflect the accumulation change of erucic acid at the position sn-2 of triacylglycerol compared with the wild type. Therefore, the gene function is verified in the arabidopsis high erucic acid type strain seeds, and the gene has important reference significance for improving the erucic acid accumulation at the triacylglycerol sn-2 position of the corresponding cruciferous oil crop.

The invention further discloses a function of the gene for facilitating the accumulation of sn-2 erucic acid in triacylglycerol in the seeds of a large oil crop cabbage type rape high erucic acid strain. The invention firstly utilizes the tropaeolum TmLPCAT gene to assemble a large amount of erucyl groups on the sn-2 position of triacylglycerol of the brassica napus, breaks the bottleneck that the erucyl groups can not be assembled on the sn-2 position of the brassica napus under the natural condition, and provides a novel method and a germplasm material for rape high erucic acid breeding.

The invention verifies the function of the gene in helping accumulation of triacylglycerol sn-2 erucic acid in novel oil crop camelina sativa seeds. The camelina sativa belongs to the Cruciferae family, has short growth cycle, strong resistance, drought and saline-alkali tolerance, and can be widely planted on barren soil. According to the invention, the camelina sativa TmLPCAT gene is used for assembling the erucyl group on the sn-2 position of the camelina sativa triacylglycerol for the first time, so that the bottleneck that the sn-2 position of the camelina sativa can not be assembled with the erucyl group almost under the natural condition of the camelina sativa is broken, and a new direction is provided for the development and utilization of the camelina sativa.

Drawings

FIG. 1 Structure of triacylglycerols and sn position of glycerol backbone; the glycerol skeleton has 3 hydroxyl groups which can be linked with fatty acid through ester bonds, and the 3 positions of the glycerol skeleton are respectively named as sn-1, sn-2 and sn-3 from top to bottom, R₁、R₂、R₃Respectively represents the hydrocarbon chain of fatty acid, the TmLPCAT gene coding enzyme of the invention can transfer fatty acyl at the sn-2 position of lysophosphatidylcholine, and further influence the type and content of fatty acid of triacylglycerol sn-2。

FIG. 2 shows the fatty acid composition (%) at triacylglycerol sn-2 position of Brassica napus and Tropaeolum plants; wherein: the abscissa indicates the fatty acid composition, C indicates the carbon chain, the numbers before the colon represent the carbon chain length, and the numbers after the colon represent the number of double bonds (e.g., C22:1 represents a fatty acid with a carbon chain length of 22 carbons and a number of double bonds of 1; C22:1 is erucic acid in the present invention); the ordinate values represent the percentage of fatty acids.

FIG. 3 shows the fatty acid composition (%) at the position of triacylglycerol sn-2 in Arabidopsis thaliana high mustard type line control (HE) and transgenic Arabidopsis plants seeds; wherein: the abscissa represents the fatty acid content, and the ordinate represents the percentage of fatty acid. The gray bars in the figure represent the percentage of fatty acids AT the triacylglycerol sn-2 position of the Arabidopsis thaliana high mustard control seed, the other bars in turn being the percentage of fatty acids AT the triacylglycerol sn-2 position of the seed of the three transgenic Arabidopsis lines AT1-AT3 in which the seed-specific Phaseolin promoter drives the TmLPCAT gene.

FIG. 4 shows the fatty acid composition (%) at the triacylglycerol sn-2 position of the seeds of high erucic acid cabbage type rape Control (Control) and transgenic plants; wherein: the abscissa represents the fatty acid content, and the ordinate represents the percentage of fatty acid.

FIG. 5 shows fatty acid components (%) at the triacylglycerols sn-2 position of camelina sativa Wild Type (WT) and transgenic camelina sativa plant seeds; wherein: the abscissa represents the fatty acid content, and the ordinate represents the percentage of fatty acid.

The present invention will be described in further detail with reference to the following drawings and examples.

Detailed Description

Unless otherwise indicated, the terms or methods herein are implemented according to conventional wisdom or known methods by one of ordinary skill in the relevant art.

In the following examples, only relatively specific research methods are described in detail. Conventional test methods and the conventional biochemical reagents involved are not described in detail, and specific reference is made to research methods such as molecular cloning, a laboratory Manual (J. SammBruk et al).

The fatty acid composition (%) at the sn-2 position of triacylglycerol described in the examples below is the percentage of fatty acids at each sn-2 position of triacylglycerol as a percentage of total fatty acids at that position.

The plant seeds used in the examples below were from laboratory-created plant test material or gift-breeding material (described below).

The first embodiment is as follows: determination of fatty acids in the sn-2 position of triacylglycerols from plant seeds

Extraction of total lipid and separation of triacylglycerol in Brassica napus and Tropaeolum

Firstly, respectively taking 50mg of cabbage type rape (a hybrid rape central inbred line in Shaanxi province) and a tropaeolum seed (purchased commercially), fully grinding and transferring the seeds into a threaded glass test tube; adding 3ml of a solution of chloroform and methanol (2:1), vortexing and shaking for 30s, and standing for 15 min; then adding 1ml of 0.9% sodium chloride solution to layer the liquid in the test tube, performing vortex oscillation again, centrifuging (1500r, 3min), taking down the clear liquid (chloroform phase) and transferring the clear liquid into another test tube, and drying the clear liquid by blowing with nitrogen, wherein the residue at the bottom of the test tube is total lipid extracted from the seeds;

dissolving the extracted total lipids in 50 μ l chloroform, and performing sample spreading on a pre-dried G60 TLC silica gel plate with n-hexane, diethyl ether and acetic acid (70:30:1), and dyeing with primrose yellow after sample spreading; naturally air-drying the TLC plate, placing the TLC plate under an ultraviolet lamp, and marking a strip corresponding to Triacylglycerol (TAG) according to a standard sample (soybean oil is diluted by 1000 times by using chloroform); the labeled TAG bands were recovered, placed in a test tube, and the reaction mixture was incubated with 600 μ l n-hexane: extracting TAG in silica gel powder with diethyl ether (1:1), repeating for 2 times, and mixing;

II, 2-MAG acquisition representing sn-2 position

(I) 1ml of the TAG sample obtained in the above step was placed in a test tube, and 0.8ml of reaction buffer (50mM borax, 5mM CaCl) was added₂pH 7.8) and 200. mu.l lipase (Novocata) in a shaker (37 ℃, 250r/min) for 4-6 h;

(II) adding 3ml of chloroform: methanol (1:1) to stop the reaction; standing for 10min after vortex oscillation, adding 1ml of 0.9% sodium chloride solution to stratify liquid in a test tube, centrifuging (1500r, 3min), taking a supernatant (chloroform phase), transferring to another test tube, drying by nitrogen, and adding 100 mu l of chloroform for dissolving;

(III) adopting an original sample-spreading solvent system n-hexane: diethyl ether: acetic acid (70:30:1), sampling the extracted product, and dyeing with primrose yellow; the stained TLC plate was placed in an ultraviolet imager, and the position of the corresponding band (divided into 4 bands, TAG, FFA, 1.2-DAG or 2.3-DAG, 2-MAG in this order from top to bottom) was determined and labeled according to the standard, and the 2-MAG band was scraped off with a razor blade and recovered, and 900. mu.l of chloroform was added: the mixture was vortexed and separated by adding 300. mu.l of 0.9% sodium chloride solution, and the supernatant was transferred to a 1.5ml gas chromatography vial and blown dry under nitrogen.

Analysis of fatty acid composition

Firstly, 200 mul of methyl esterification reaction liquid (containing 5 percent of concentrated H) is added into a 1.5ml gas phase sample injection bottle₂SO₄CH (A) of₃OH solution) is added into the mixture, and water bath is carried out for 2h at the temperature of 85 ℃; taking out the sample injection bottle after the reaction, standing, cooling to room temperature, and adding 200 μ l of 0.9% NaCl solution to stop the reaction; adding 100 μ l n-hexane, mixing well, centrifuging at 1500rpm for 10min at normal temperature; taking the supernatant, placing the supernatant in a liner tube rinsed by n-hexane, and measuring fatty acid components;

and (II) analyzing FAMEs by adopting a capillary chromatographic column DB-23(Agilent, 30m), wherein the gas chromatographic analysis parameters are as follows: the initial temperature of the capillary column box is 50 ℃, the capillary column box is kept for 60s, then the temperature is raised to 175 ℃, the temperature raising rate is 35 ℃/min, and the capillary column box is kept for 60 s; heating to 230 deg.C, heating at a rate of 4 deg.C/min, and maintaining for 5 min; hydrogen flow rate of 40ml/min, air flow rate of 40ml/min, carrier gas flow split ratio N₂:H₂10: 1; the amount of sample was 4. mu.l.

The fatty acid composition analysis of sn-2 position in brassica napus seeds and tropaeolum majus seeds is shown in figure 2.

Example two: functional analysis of TmLPCAT gene at sn-2 site of triacylglycerol in Arabidopsis thaliana seeds

Cloning of TmLPCAT Gene

Firstly, the tropaeolum embryo which blooms for 25 days is processed according to Trizol^TMTotal RNA was extracted using the procedure provided in kit (Invitrogen) and the DNA fragment containingPrimeScript of gDNA digestive enzyme^TMRT kit (Takara) is reverse transcription into cDNA, and the operation is carried out on ice;

and (II) excavating transcriptome data of developed tropaeolum seeds, designing a primer according to the screened tropaeolum LPCAT transcript, and performing amplification reaction on the transcriptome data by adopting a TmLPCAT-F: 5'-ATGGACCTCGACATGGAA-3' and TmLPCAT-R: 5'-TCACTGTTCTTTTCTCGCTT-3' as a primer, and using the cDNA obtained above as a template to amplify the coding region of the TmLPCAT gene;

and (III) connecting the sequence with a cloning vector and sequencing for identification. The amplified fragment was cloned into pUC57 entry vector containing attL1 and attL2 and the E.coli of the fragment of interest was sequenced and the sequencing result of the fragment of interest is shown in SEQ ID NO. 1.

Second, plant seed specific expression vector construction and agrobacterium preparation

The vector used in this example was Pha-DsRed-pK7WG2D, constructed in the laboratory of the applicant, and the vector backbone was the universal expression vector pK7WG2D, in which the 35S promoter carried by it was replaced with the seed-specific Phaseolin promoter (Pha), and the DsRed fluorescent protein marker gene expression cassette was ligated.

By the LR enzyme (A)

LR

II Mix) catalyzing the reaction of the entry carrier and the target carrier, wherein the reaction conditions are as follows: reacting at 25 ℃ for 1h to obtain an expression vector Pha-TmLPCAT-DsRed-pK7WG2D containing a TmLPCAT sequence;

mu.l of the expression vector was added to 20. mu.l of Agrobacterium-infected cells (GV3101, purchased from Vital organisms), mixed well and allowed to stand on ice for 5min, then snap frozen for 5min with liquid nitrogen, taken out, washed in water at 37 ℃ for 5min, and then placed on ice for 2 min. Adding 500 μ l culture medium, culturing in 28 deg.C shaking table for 4-6 hr, uniformly coating on solid culture medium containing corresponding antibiotics, identifying with TmLPCAT-F/R primer after bacterial colony grows out, culturing to obtain correct strain, and storing for use. As an alternative to this embodiment, the above-described expression vector may also be added to E.coli.

Transformation of Arabidopsis thaliana high mustard lines

Transformation of Arabidopsis thaliana high-mustard line (HE, created by The Applicant' S laboratory) seeds were harvested after cultivation for about 3 weeks using The inflorescence dip transformation method (Clough S J, Bent A F. floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana [ J ]. The plant journel, 1998,16(6):735-743), by culturing The Agrobacterium containing The expression vector at 28 ℃ until The spectroscopic value of OD600 reached between 0.8 and 1.0, centrifuging at 4000r/min for 10min, discarding The supernatant, resuspending it with 5% sucrose solution and adding 0.05% Silwet L-77, after The inflorescence dip of The Agrobacterium, and culturing.

And (II) selecting transgenic positive seeds by using fluorescence labeling protein. Since the fluorescent marker protein DsRed and the target gene are both constructed on the expression vector and are expressed in the plant body, the positive seeds emitting red fluorescence can be picked out by observing through a red filter under the green light with the wavelength of 543 nm.

Fourth, triacylglycerol sn-2-bit erucic acid analysis of transformed strain seed

The selection transformation yielded 3 homozygous transformation lines, designated AT1, AT2, AT3, respectively. After the seeds are harvested, fatty acid components at the sn-2 position of triacylglycerol are measured by the method of the first embodiment, and the results of the percentage of erucic acid contained at the sn-2 position are shown in the attached figure 3. The result shows that the erucic acid AT the sn-2 position of the triacylglycerol of the TmLPCAT Arabidopsis transformed line seed is obviously accumulated, the erucic acid is increased to more than 1 percent from 0.3 percent in the wild type, the line AT1 which is increased most is increased to 3.8 percent, and the increase amplitude is more than 10 times.

Example three: application of TmLPCAT in cabbage type rape and camelina sativa

First, cabbage type rape transformation

The cabbage type rape seed LC (erucic acid content is about 50%, selfing line bred by hybrid rape center in Shaanxi province) is sterilized by tissue culture method, sowed on culture medium, and dark cultured for 5 days at 25 deg.C. Cutting the hypocotyl of the germinated rape seedling, wherein the length of the hypocotyl is about 1 cm; placing the cut explant into an agrobacterium liquid culture dish containing the TmLPCAT expression vector after activation, and infecting for 15 min; transferring to callus induction phase culture medium (CIM), dark culturing at 25 deg.C for two days, and culturing under light; transferring the seeds into a germination induction culture medium (SIM) after 3 weeks, and carrying out subculture once every 2 weeks until green buds appear; transferring into root inducing culture medium (RIM) after germination, and culturing for 2-4 weeks to root.

(II) selecting transgenic positive seeds by using fluorescence labeling protein, observing fluorescence in cotyledons of rape seeds so as to select positive strains, and selecting 3 rape transgenic T₂Strains, respectively named BN1-3, and seeds are used for determining fatty acid components.

(III) triacylglycerol sn-2-bit erucic acid analysis of transformed line seeds

The percentage of erucic acid in the sn-2 position of triacylglycerol was determined by the method described in the above example and shown in FIG. 4. The result shows that a large amount of erucic acid is accumulated at the sn-2 position of triacylglycerol of the TmLPCAT brassica napus transformation strain seeds, the erucic acid is improved to more than 2.8 percent from 0.5 percent of the contrast, and the strain BN2 which is improved most is improved to nearly 6 percent and is improved by nearly 12 times.

II, transformation of camelina sativa

The transformation of the camelina sativa variety Suneson (present at Li university of Monda, USA) adopts an inflorescence dipping transformation method. Agrobacterium containing the expression vector (GV3101) was cultured at 28 ℃ until the spectroscopic OD600 reached between 0.8 and 1.0, centrifuged at 4000r/min for 10min, the supernatant was discarded, resuspended in 5% sucrose solution and 0.05% Silwet L-77 was added. The seeds can be harvested after the inflorescences are dipped and cultured for about 5-6 weeks.

(II) selecting transgenic positive seeds by using fluorescence labeling protein

Selection of transformation to obtain transformation T₂Lines, non-transgenic seeds, transgenic seeds and wild-type seeds were rejected on the basis of red fluorescence for determination of fatty acid composition.

(III) triacylglycerol sn-2-bit erucic acid analysis of transformed line seeds

The percentage of erucic acid contained in the triacylglycerol sn-2 position of the wild type and transformed lines was determined using the method described in example one. The results show that the erucic acid at the sn-2 position of triacylglycerol in the seeds of TmLPCAT camelina sativa transformed line is only increased in one of the tested lines, which may be related to lower background content of erucic acid in camelina, and the erucic acid accumulation amount is about 1.1% in the transgenic line with increased erucic acid, and is increased by about 63.8% compared with 0.7% of wild type, as shown in FIG. 5. WT and CS in FIG. 5 represent camelina wild-type (Suneson) and TmLPCAT transgenic lines, respectively.

Nucleotide sequence list electronic file

<110> northwest agriculture and forestry science and technology university

Application of <120> TmLPCAT gene in improving content of triacylglycerol sn-2 bit ultra-long chain fatty acid

<141>

<160>

<210>1

<211>1398

<212>DNA

<213> TmLPCAT Gene

<220>

<223>

<400>1

1 ATGGACCTCG ACATGGAATC CATGGCGGCC ACGATCGGGG TCTCCGTCTC GGTCCTAAGG

61 TTCCTTCTCT GTTTCGGGGC TACGATTCCC GTTTCTTTCA TATGGAGACT CGTTCCAGGT

121 CGACTCGCTA AGCATCTCTA CGCGGCTGGG TCAGGAGTCT TCCTCTCGTA TCTATCCTTT

181 GGGTTTTCTT CAAATCTTCA TTTCCTGGTG CCTATGATTC TGGGTTACGC CTCCATGGTG

241 TTGTATCGTC GGAAGTGTGG TATAATAACG TTTTTGTTGG GATTCGGATA TCTTATTGGC

301 TGCCATGTAT ATTACATGAG TGGGGATGCG TGGAAGGAAG GAGGAATTGA TGCAACTGGA

361 GCCTTGATGG TTTTAACGCT TAAAGTCATC TCGTGTGCTA TAAATTACAA TGATGGATTA

421 CTGAAAGAGG AAGAAAGCTT ACGTGAATCA CAAAAGAAAA ATAGGTTGAT GAAGTTACCT

481 TCCATTCTTG AGTATTTTGG TTACTGTCTC TGCTGTGCTA GTCACTTTGC TGGTCCGGTT

541 TATGAAATGA AGGATTATCT TGACTGGACC GAAGGGAAAG GGATTTGGGC TCCTAATAAG

601 AATGGGCGAT CACCCTCACC TTATGGGGCT ACAATTCGTG CTATTTTTCA AGCTGCTATT

661 TGCATGGCTT TGTATCTTTA CCTAGTACCC TACCATCCCT TATCCAGATT TACTGACCCT

721 ATATACCAAC AATGGGGATT CTGGAGACGG TTGAGCTATC AATACATGTC TGGCTTTACA

781 GCACGCTGGA AATATTATTT TATCTGGTCG ATTTCAGAGG CCTCTGTCAT TATTTCTGGC

841 CTGGGTTTCA GTGGTTGGAC AGATTCTTCC CCACCAAAGC CACGCTGGGA CCGGGCAAAA

901 AATGTAGACA TTTTCGGCGT TGAGCTCGCA AAGAGTGCTG TTCAGTTGCC ACTTGTCTGG

961 AACATACAAG TCAGCACTTG GCTACGTCAC TATGTTTATG ACAGACTTGT TAAGAAAGGA

1021 ACGAAAGCAG GGTTCTTCCA GTTGCTGGCT ACACAGACTG TCAGTGCAGT TTGGCATGGT

1081 CTATATGCTG GGTATATCAT ATTCTTTGTT CAGTCCGCAC TGATGATTGC CGGTTCAAAA

1141 ACCATTTACA GATGGCAACA AGCTGTTCCT GCAAATGCGG CTCTTGTCAA AAAAGTGCTG

1201 GTTATCATGA ACTTTTTGTA CACTATTTTG GTTCTAAACT ACTCCTGCGT TGGGTTTATG

1261 GTGCTAAGCT TGCATGAAAC CCTTACCTCA TATAAGAGCG TGTATTTTAT TGGAACAGTT

1321 ATACCTGTAG TCGTGATTCT CCTTGGTTAC ATAATACCTG CAAAGCCAGC AAGATCGAAA

1381 GCGAGAAAAG AACAGTGA

<210>2

<211>465

<212>PROTEIN

<213> enzyme encoded by TmLPCAT Gene

<220>

<223>

<400>2

1 MDLDMESMAA TIGVSVSVLR FLLCFGATIP VSFIWRLVPG RLAKHLYAAG SGVFLSYLSF

61 GFSSNLHFLV PMILGYASMV LYRRKCGIIT FLLGFGYLIG CHVYYMSGDA WKEGGIDATG

121 ALMVLTLKVI SCAINYNDGL LKEEESLRES QKKNRLMKLP SILEYFGYCL CCASHFAGPV

181 YEMKDYLDWT EGKGIWAPNK NGRSPSPYGA TIRAIFQAAI CMALYLYLVP YHPLSRFTDP

241 IYQQWGFWRR LSYQYMSGFT ARWKYYFIWS ISEASVIISG LGFSGWTDSS PPKPRWDRAK

301 NVDIFGVELA KSAVQLPLVW NIQVSTWLRH YVYDRLVKKG TKAGFFQLLA TQTVSAVWHG

361 LYAGYIIFFV QSALMIAGSK TIYRWQQAVP ANAALVKKVL VIMNFLYTIL VLNYSCVGFM

421 VLSLHETLTS YKSVYFIGTV IPVVVILLGY IIPAKPARSK ARKEQ

Claims

1. Herb of Manyflower IrisLPCATUse of a gene for increasing erucic acid content on the sn-2 position of triacylglycerols in plants, said tropaeolum majusLPCATThe gene is a sequence shown in SEQ ID NO. 1.

2. Comprises the eclipta alba of claim 1LPCATExpression vector of gene.

3. The expression vector of claim 2, wherein the expression vector is Pha-TmLPCATDsRed-pK7WG2D, where Pha is the Phaseolin promoter,TmLPCATthe eclipta alba of claim 1LPCATThe gene DsRed is a fluorescent protein marker gene expression cassette, and pK7WG2D is an expression vector framework.

4. Comprises the eclipta alba of claim 1LPCATA bacterial cell of the gene.

5. The cell of claim 4, comprising the Tropaeolum majus of claim 1LPCATGenetically Escherichia coli or Agrobacterium cells.

6. Herb of Manyflower IrisLPCATUse of a gene encoded enzyme for increasing the content of erucic acid in the sn-2 position of triacylglycerols in plants, said tropaeolum majusLPCATThe enzyme coded by the gene is a sequence shown in SEQ ID NO. 2.

7. The use according to claim 1 or 6, wherein the plant is an oil crop.

8. The use according to claim 7, wherein the oil crop plant is selected from Arabidopsis thaliana, Brassica napus or camelina sativa.