CN110257444B - Method for producing medium-chain fatty acid in plant cells - Google Patents

Abstract

The invention discloses a method for producing medium chain fatty acid in plant cells, which adopts a Cre/LoxP system to successfully construct three polygene systems of fatB3-LPAAT, fatB3-KASI and fatB3-LPAAT-KASI and then transfers the polygene systems into Arabidopsis to carry out seed-specific co-expression, comprising the following steps: constructing a polygene coexpression vector, preparing agrobacterium competence, transforming the polygene coexpression vector into agrobacterium competence, impregnating arabidopsis thaliana by using an agrobacterium dip method, verifying transgenic arabidopsis thaliana, performing fluorescence quantitative PCR on arabidopsis thaliana seeds, extracting fatty acid of the arabidopsis thaliana seeds, and performing GC measurement. The three polygene systems of FatB3-LPAAT, fatB3-KASI and FatB3-LPAAT-KASI are successfully constructed by adopting the Cre/LoxP system, and are transferred into Arabidopsis thaliana for seed-specific co-expression, so that the co-expression trend of three genome combinations is analyzed, and the content of synthesized medium chain fatty acid in plant cells is effectively improved.

Description

Method for producing medium-chain fatty acid in plant cells

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for producing medium-chain fatty acid in plant cells.

Background

Medium chain fatty acids generally refer to fatty caproic acid with carbon element 6 to lauric acid with carbon element 12. Medium chain fatty acid is mainly decomposed by lipase in human body and transported to liver through blood vessel to carry out beta oxidation to release energy, and the metabolism speed is 10 times that of long chain fatty acid, so that obesity is not easy to form. Meanwhile, the medium-chain fatty acid affects the cell signal intensity in cells by regulating the cell metabolism, and in addition, compared with common animal and plant oil, the medium-chain fatty acid has the advantages of higher oxidation stability, small viscosity, small surface tension, excellent dissolving capacity for various compounds and the like, and can be used as a good solvent for various medicines such as vitamins, antibiotics and the like.

The natural medium-chain fatty acid is generally from animal milk products, the rest is mainly from extraction of oil palm and coconut, the source is limited, the extraction of the medium-chain fatty acid in the oil palm and the coconut is complex, and the extraction amount is small; the plant genetic engineering can directionally improve the oil content of plants so as to improve the oil yield of unit area, and the total yield of medium-chain fatty acid can not be improved by enlarging the planting area due to limited jointly available land. At present, a method for producing medium-chain fatty acid by using a genetic engineering means has not been reported yet.

Therefore, it is a problem to be solved by those skilled in the art how to provide a method for producing medium-chain fatty acids and improving the total yield of medium-chain fatty acids by genetic engineering means.

Disclosure of Invention

In view of this, the present invention provides a method for producing medium-chain fatty acids in plant cells, which uses genetic engineering means to construct FATB3, LPAAT and KASI polygene coexpression vectors and transfer them into arabidopsis thaliana, in which medium-chain fatty acids are produced, increasing the yield of medium-chain fatty acids.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a method for producing medium chain fatty acids in plant cells, which successfully constructs three polygenic systems of FatB3-LPAAT, fatB3-KASI or FatB3-LPAAT-KASI using Cre/LoxP system and transfers them into arabidopsis for seed-specific co-expression, comprising the following steps:

constructing a polygene coexpression vector, preparing agrobacterium competence, transforming the polygene coexpression vector into agrobacterium competence, impregnating Arabidopsis thaliana by using an agrobacterium dip method, verifying transgenic Arabidopsis thaliana, performing fluorescence quantitative PCR on Arabidopsis thaliana seeds, extracting fatty acid of Arabidopsis thaliana seeds, and performing GC determination.

Optionally, the construction of the polygene coexpression vector comprises the following steps:

1) Constructing a donor vector; 2) Constructing a polygene coexpression vector by using FatB3, LPAAT and pYLTAC380DTH plasmids; 3) Constructing a polygene coexpression vector by using FatB3, KASI and pYLTAC380DTH plasmids; 4) The multigene co-expression vector was constructed with the FatB3, LPAAT, KASI, and pyl tac380DTH plasmids. Wherein the sequence of the FatB3 gene is shown in SEQ ID No. 1; the LPAAT gene sequence is shown as SEQ ID NO. 2; the KASI gene sequence is shown as SEQ ID NO. 3.

Optionally, the construction of the donor vector comprises:

1) Constructing Napin-fatB3-d1/d2 plasmids by Napin, fatB3 and pYL322-d1 or Napin, fatB3 and pYL322-d2 plasmids respectively, and carrying out mixed electric shock on the Napin-fatB3-d1/d2 plasmids and pYLTAC380DTH plasmids to transfer the mixture into escherichia coli competent NS3529 capable of generating Cre recombinase, and after the NS3529 is activated, coating the mixture in LB solid culture medium added with Kana+ and Cm+ for expansion culture, and culturing the mixture at 37 ℃ for 22h;

2) Extracting plasmid in NS3529 after amplification, performing I-SceI digestion on the plasmid, recovering the digested fragments, transferring the fragments into escherichia coli competent TransT1 by heat shock, coating the fragments on an X-gal medium added with Kana+ after the TransT1 is activated, culturing the fragments for 12 hours at 37 ℃, selecting white colonies for colony PCR, and screening positive colonies;

3) Amplifying and culturing the screened positive colony, and extracting the plasmid in the amplified and cultured TransT1 bacterial liquid to obtain the FatB3-380DTH plasmid.

Optionally, the constructing method of the polygene coexpression vector in the step 2) comprises the following steps:

1) The 5400-LPAAT-d1/d2 plasmid was constructed with the promoters 5400, LPAAT and pYL322-d1 or with the promoters 5400, LPAAT and pYL322-d2 plasmids, and the 5400-LPAAT-d1/d2 and fatB3-380DTH plasmids were used as follows: 1-2:1, in the competent NS3529 of the electric shock transformed escherichia coli, after the NS3529 is activated, the strain is coated in an LB solid medium coated with Kana+ and amp+ for expansion culture, and is cultured for 22 hours at 37 ℃;

2) Extracting plasmid in NS3529 after amplification, performing I-SceI digestion on the plasmid, recovering the digested fragments, transferring the fragments into escherichia coli competent TransT1 by heat shock, coating the fragments on an X-gal medium added with Kana+ after the TransT1 is activated, culturing the fragments for 12 hours at 37 ℃, selecting white colonies for colony PCR, and screening positive colonies;

3) Amplifying and culturing the screened positive colony, and extracting plasmids in the amplified and cultured TransT1 bacterial liquid to obtain the FatB3-LPAAT-380DTH polygene coexpression vector.

Alternatively, the construction of the polygene coexpression vector in the step 3) is to construct the promoters 16460, KASI and pYL-322-d 1 or the plasmids 16460, KASI and pYL-322-d 2 into 16460-KASI-d1/d2 according to the construction method of the fatB3-LPAAT-380DTH polygene coexpression vector, and to construct the fatB3-KASI-380DTH polygene expression vector by recombination of the plasmids 16460-KASI-d1/d2 and the fatB3-380 DTH.

Optionally, the construction method of the polygene coexpression vector in the step 4) is to reconstruct 16460-KASI-d1/d2 and fatB3-LPAAT-380DTH to construct the fatB3-LPAAT-KASI-380DTH polygene expression vector.

Optionally, the agrobacteria dip-dyeing arabidopsis thaliana comprises the following steps:

1) Transferring into agrobacterium with three polygene systems including FatB3-LPAAT, fatB3-KASI or FatB3-LPAAT-KASI, streaking the transformed agrobacterium liquid on a plate containing 50ug/mL Rif+ and 50ug/mL kana+ antibiotics, and culturing at 28 ℃ for 2-3 days;

2) Picking up single colony for colony PCR, selecting positive colony, inoculating into 20mL liquid culture medium coated with Rif+ and Kana+, shake culturing at 28deg.C and 200rpm to OD ₆₀₀ ＝1.0；

3) Preparing agrobacterium infection liquid according to a proportion, adding 25g of sucrose, 0.25g of Silwet7750 mu L of MES acid and 1.05g of MS powder into 500mL of the infection liquid, and supplementing to 500mL by using sterile water;

4) Selecting the period with the most inflorescences of the wild arabidopsis, cutting off the grown fruit clamp, and immersing the inflorescences in the soaking solution for 3min;

5) Placing Arabidopsis thaliana in a basin horizontally after dip dyeing is finished, culturing in the dark for 24 hours, and repeating dip dyeing after one week; after the dip dyeing is finished, the arabidopsis thaliana is normally placed and cultivated to seed.

Alternatively, the kit used for PCR positive identification of colony and transgenic plant DNA is 2xF8FastLong PCRMasterMix, and the system is as follows:

the amplification system is as follows:

alternatively, the Primer sequences of the three genes and the Primer sequence of the promoter are designed by using Primer premier6.0, and the specific sequences are as follows:

the sequence of the positive primer of FATB3 is shown as SEQ ID NO. 4; the reverse primer sequence is shown as SEQ ID NO. 5; the forward primer sequence of the LPAAT is shown as SEQ ID NO. 6; the reverse primer sequence is shown as SEQ ID NO. 7; the forward primer sequence of KASI is shown as SEQ ID NO. 8; the reverse primer sequence is shown as SEQ ID NO. 9; the forward primer sequence of At16460 is shown in SEQ ID NO. 10; the reverse primer sequence is shown as SEQ ID NO. 11; the forward primer sequence of At5400 is shown as SEQ ID NO. 12; the reverse primer sequence is shown as SEQ ID NO. 13; the forward primer sequence of NAPIN is shown as SEQ ID NO. 14; the reverse primer sequence is shown in SEQ ID NO. 15.

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Alternatively, a Primer premier6.0 is used for designing a fluorescence quantitative Primer according to three gene sequences, a conserved gene beta-actin is used as an internal reference Primer, a takaRa fluorescence quantitative kit is used for real-time fluorescence quantitative, and the Primer sequences are as follows:

the forward primer sequence of RT-FATB3 is shown as SEQ ID NO.16, and the reverse primer sequence is shown as SEQ ID NO. 17; the forward primer sequence of RT-KASI is shown as SEQ ID NO.18, and the reverse primer sequence is shown as SEQ ID NO. 19; the forward primer sequence of RT-LPAAT is shown as SEQ ID NO.20, and the reverse primer sequence is shown as SEQ ID NO. 21; the forward primer sequence of the beta-actin is shown as SEQ ID NO.22, and the reverse primer sequence is shown as SEQ ID NO. 23.

Alternatively, the PCR system for double digestion of the target fragment and the vector is as follows:

the PCR cycle conditions were: 37 ℃,30min, 65 ℃,10min, 4 ℃ and infinity.

Alternatively, in the construction of the polygenic co-expression vector, the recovery and purification of the fragment of interest and the vector are performed according to the Gel Extraction Kit (OMEGA) kit procedure.

Alternatively, when constructing the polygene coexpression vector, the PCR system for connecting the target fragment and the vector is as follows:

the PCR cycle conditions were: 22 ℃,60min, 4 ℃ and infinity.

Optionally, the NS3529 competent preparation method is:

(1) NS3529 was streaked on antibiotic-free LB plates and incubated overnight at 37 ℃.

(2) Single colonies were picked up and cultured in 8-10mL SOB medium for 8-12h at 1:100 proportion of the culture medium is inoculated into 100mL of SOB culture medium for shake culture for 2-3h to OD ₆₀₀ The value is 0.3-0.4.

(3) Placed on ice for 10min, sub-packed into 2 50mL sterile centrifuge tubes and centrifuged at 4000rpm for 10min at 4 ℃.

(4) Discard supernatant, centrifuge tubeAdding a small amount of ddH ₂ O, light suspension precipitation, and ddH precipitation ₂ O was added to 50mL and centrifuged at 4000rpm at 4℃for 10min.

(5) Repeating (4) once.

(6) Carefully discard the supernatant, add a small amount of pre-chilled sterile 15% glycerol, re-suspend the cells, top up to 50mL, centrifuge at 4000rpm for 10min at 4deg.C, discard the supernatant.

(7) The thalli are resuspended by 2-3mL of 15% sterile glycerol, and the thalli are split into 1.5mL sterile EP pipes for standby according to the equal volume of 100uL or are rapidly frozen by liquid nitrogen and then are put into a refrigerator at the temperature of minus 80 ℃ for standby.

Compared with the prior art, the invention discloses a method for producing medium-chain fatty acid in plant cells by using a genetic engineering means, wherein a two-gene co-expression FatB3-LPAAT system, a two-gene co-expression FatB3-KASI system and a three-gene co-expression FatB3-LPAAT-KASI recombinant vector are respectively constructed by using a Cre/loxP multi-gene vector recombination technology, and are respectively transferred into wild arabidopsis thaliana for seed-specific over-expression, and the co-expression synergistic effect of the three genes in the medium-chain fatty acid grease synthesis process is further verified by detecting the expression condition of the genes and the composition and content of transgenic seed fatty acid. Wherein the promoter of FatB3 is a seed specific promoter Napin, and the promoters of LPAAT and KASI are promoters At5400 and At16460, respectively, specifically expressed in arabidopsis seed embryo and endosperm; the terminators of the three genes are all terminator NOS derived from a plant expression vector pCAMBIA 1300S. The sequence of pCAMBIA1300S is shown in SEQ ID NO. 24.

The analysis result of the T3 generation homozygote fatty acid of the transgenic arabidopsis thaliana shows that:

1. seed from fatB3-LPAAT, fatB3-KASI and fatB3-LPAAT-KASI transgenic plants produced a C12:0 (relative percentage) increase of at least 395%, 124% and 134% over wild type; similarly, C14:0 of seed of FatB3-LPAAT, fatB3-KASI and FatB3-LPAAT-KASI transgenic plants was also increased by at least 383%, 102% and 106% over wild type seed; other species of fatty acids did not show significant differential changes.

2. The seed of the FatB3-LPAAT transgenic plant produced about 92% and 58% more c14:0 (relative percentage) than the seed of the FatB3-LPAAT transgenic plant and the seed of the FatB3-LPAAT-KASI transgenic plant, respectively, and similarly, the seed of the FatB3-LPAAT-KASI transgenic plant produced about 19% and 116% more c12:0 than the seed of the FatB3-LPAAT and the seed of the FatB3-KASI transgenic plant. The results show that co-expression of coconut FatB3 and LPAAT in plants is better than co-expression of the three genes FatB-LPAAT-KASI and two genes FatB3-LPAAT to produce medium chain fatty acids.

3. Analysis of the total content of medium chain fatty acids in transgenic seeds found that FatB3-LPAAT, fatB3-KASI and FatB3-LPAAT-KASI transgenic plant seeds increased at least 47%, 70% and 56% respectively over wild type seeds, while the total content of medium chain fatty acids was not significantly different among the three transgenic lines. The results show that: in the synthesis and accumulation of medium chain fatty acids in coconut, coconut FatB3 and LPAAT play a major role, whereas coconut KASI does not play a very significant role in either altering the fatty acid composition or increasing the total fatty acid content.

In conclusion, three polygene systems of FatB3-LPAAT, fatB3-KASI and FatB3-LPAAT-KASI are successfully constructed by adopting the Cre/LoxP system in the experiment, and are transferred into Arabidopsis thaliana to perform seed-specific co-expression, so that the co-expression trend of three genome combinations is analyzed, and the synthesis amount of medium chain fatty acid in plant cells is improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the cleavage sites of two vectors pYL-d 1 and pYL-322-d 2 of the present invention;

FIG. 2 is a schematic diagram of the receptor vector pYLTAC380DH of the present invention;

FIG. 3 is a schematic diagram showing three target genes and promoter cleavage sites corresponding to the target genes;

FIG. 4 is a schematic diagram of a donor vector construction process according to the present invention;

FIG. 5 is a schematic diagram showing colony PCR of promoters Napin, 16460 and gene KASI of the present invention;

FIG. 6 is a schematic diagram showing colony PCR of the promoter 5400, genes LPAAT and fatB3 of the present invention;

FIG. 8-1 is a graph showing the cleavage results of FatB3-380DTH NotI;

FIG. 8-2 shows the results of the digestion of FatB3-LPAAT-380DTH, fatB3-KASI, fatB3-LPAAT-KASI-380DTH NotI;

FIG. 8 is a schematic diagram of single gene positions in a multigenic vector;

FIG. 9 is a schematic representation of positive plants screened for hygromycin;

FIG. 10 is a drawing showing transgenic Arabidopsis thaliana T ₂ Generation PCR positive identification schematic diagram;

FIG. 11 is a graph showing the fluorescence quantification results of three genes FatB 3-LPAAT-KASI;

FIG. 12 is a graph showing the fluorescence quantification result of two genes FatB 3-LPAAT;

FIG. 13 is a graph showing the fluorescence quantification results of two genes FatB 3-KASI;

FIG. 14 is a graph showing the results of fatty acid methyl esters of the fatB3-LPAAT-KASI three genes;

FIG. 15 is a graph showing fatty acid methyl ester results for the fatB3-LPAAT two genes;

FIG. 16 is a graph showing fatB3-KASI two-gene fatty acid methyl ester results;

FIG. 17 is a graph showing comparison of fatty acid results of 17 lines in FLK, FK line 9 and 34 lines in FL;

FIG. 18 is a diagram showing total fatty acid methyl esters of the three genes fatB3-LPAAT-KASI and the two genes fatB3-LPAAT and fatB 3-KASI.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

In the invention, wild arabidopsis seeds, escherichia coli Trans1-T1 competent cells and agrobacterium tumefaciens strain GV3101 are all preserved in the laboratory. The Cre/LoxP multiple genome transformation and transformation vector system is provided by the Proprietary of agricultural university of North China Liu Yaoguang, which comprises two vectors pYL-d 1 and pYL-d 2, one receptor vector pYLTAC380DTH and an E.coli strain NS3529 expressing Cre recombinase, and FIG. 1 shows the available cleavage sites of vectors pYL-d 1 and pYL-d 2, and FIG. 2 is a schematic diagram of the receptor vector pYLTAC380 DTH.

This example discloses the formation of a donor vector, and in the early stages of the experiment, the inventors have cloned three genes, fatB3 (GenBank Accession: JF 338905.1), KASI (GenBank Accession: JX 275887), LPAAT (genbankcession: xp_ 002522947.1) into pCAMBIA1300S vector.

Wherein the sizes of FatB3, KASI and LPAAT are 1245bp, 1485bp and 927bp respectively, the promoter Napin is a seed-specific expression promoter on a vector pCAMBIA1300S, the size is 361bp, fatB3 is expressed in the process of seed maturation, the promoter 16460 expresses KASI in the process of seed endosperm formation, the size is 882bp, and the promoter 5400 is the promoter for the initiation of expression of LPAAT in seed endosperm and inner beads, and the size is 716bp.

The restriction sites of the gene and its promoter were designed according to the restriction sites of the 1300S vector and the restriction sites of the donor vector as shown in FIG. 3, wherein the terminators of the three modules were derived from the terminator NOS on pCAMBIA 1300S. The map of pCAMBIA1300S is shown in fig. 4; since the promoter of FatB3 is Napin, the 1300S plasmid with the Napin promoter, pYL-d 1 and pYL-d 2 plasmid are firstly digested by HindIII and KpnI, then the target fragment and the target vector are recovered, then the Napin promoter is cloned into pYL-d 1 or pYL-322-d 2 vector to obtain Napin-d1/d2, colony PCR is used for positive identification, subsequent experiments are carried out after no mutation is detected in the sequencing, the similar method is used for cloning the gene FatB3 into Napin-d1/d2 by using endonucleases BamHI and EcoRI to form a donor vector Napin-FatB3-d1/d2, and the subsequent experiments are carried out after sequencing. The remaining two genes and promoters form an expression module as shown in FIG. 5.

Three expression modules are positively identified by colony PCR, wherein electrophoresis channels 3 and 5 are the promoter 16460 of 16460-KASI-d1 and the colony PCR amplification result of the gene KASI in FIG. 6, electrophoresis channels 4 and 6 are the colony PCR amplification results of the promoter 16460 for 16460-KASI-d2 and the colony PCR amplification result of the gene KASI, and electrophoresis channels 1 and 2 are the colony PCR amplification results of the promoter Napin of Napin-fatB3-d1/d2 respectively; in FIG. 7, electrophoresis channels 3 and 5 are 5400-LPAAT-d1 (construction method is the same as that of Napin-fatB3-d1/d 2) promoter 5400 and gene LPAAT colony PCR amplification result thereof, and electrophoresis channels 4 and 6 are 5400-LPAAT-d2 promoter 5400 and gene LPAAT colony PCR amplification result thereof; electrophoresis channels 1 and 2 are respectively the PCR amplification results of the colony of the gene FatB3 of Napin-FatB3-d1/d 2. Through sequencing verification, 6 expression modules, namely Napin-FatB3-d1/d2, 5400-LPAAT-d1/d2 and 16460-KASI-d1/d2 (the construction method is the same as that of Napin-FatB3-d1/d 2) are obtained in total.

EXAMPLE 2 construction of the polygene vector

The Napin-FatB3-d1 plasmid (about 400 ng) and pYLTAC380DTH plasmid (about 1 μg) are mixed and shocked into the escherichia coli competent NS3529 capable of generating Cre recombinase, activated and then coated with LB plates (Kana+, cm+), cultured for 22h at 37 ℃, plasmids are extracted by using sterile washing plates (with the colony number being more than 200), subjected to digestion by using I-SceI, recovered and heat shock and transferred into escherichia coli competent TransT1, activated and then coated with X-gal medium (Kana+), cultured for 12h at 37 ℃, white colonies are selected for colony PCR, positive colony amplification is obtained, meanwhile, verification is carried out by using LB plates (Cm+), sterile colony growth is carried out, verification is carried out by using NotI digestion plasmids, electrophoresis channel 1 is 380DTH vectors I digestion results, electrophoresis channel 2 is FatB3-380DTH vectors NotI digestion results, and subsequent experiments are carried out after sequencing.

5400-LPAAT-d2 plasmid (about 400 ng) and fatB3-380DTH plasmid (about 1 μg) are subjected to electric shock transformation in escherichia coli competent NS3529 according to a proportion, activated and then coated with LB plate (Kana+ Amp+), cultured for 22h at 37 ℃, extracted with sterile washing plate (colony number is more than 200), digested with PI-SceI, recovered and heat-shock transferred into escherichia coli competent TransT1, activated and then coated with X-gal medium (Kana+), cultured for 12h at 37 ℃, white colonies are selected for colony PCR, positive colony expansion is obtained and verified by using LB plate (amp+), the plates are free of colony growth, and verified by using NotI digested plasmid, electrophoresis channel 1 in FIG. 8-1 is 380DTH vector I digested result, electrophoresis channel 2 is FatB 3-LPAAT-380H (FL) vector I digested result, and subsequent experiments are performed after sequencing without error.

16460-KASI-d2 and fatB3-380DTH according to the second round of polygene recombination system, the polygene vector fatB3-KASI (FK for short) was obtained, no colony growth was verified by using ampicillin LB plate and no colony growth was verified by using NotI cleavage, the electrophoresis channel 3 in FIG. 8-1 was the result of fatB3-KASINOTI cleavage, and subsequent experiments were performed after sequencing verification.

16460-KASI-d1 and fatB3-LPAAT-380DTH were carried out according to the first multiplex system to obtain the polygenic vector fatB3-LPAAT-KASI-380DTH, which was verified by ampicillin LB that no colony was grown and NotI cleavage was used, and FIG. 8-1 shows that electrophoresis channel 4 was the result of NotI cleavage of fatB3-LPAAT-KASI (FLK for short), which was sequenced and verified for the subsequent experiments.

The relative positions of the single genes in the three polygene vectors were constructed using DNAMAN, as shown in fig. 9, with hygromycin selection markers in the polygene vectors, wherein FatB3 in FL, FK polygene vector system was oriented opposite to LPAAT, KASI, fatB3 in FLK was oriented the same as KASI, opposite to LPAAT, but the orientation of the genes in the polygene vector did not affect gene expression.

Example 3 Agrobacterium-mediated exhaust Arabidopsis thaliana and positive detection

The method comprises the steps of performing electric agrobacterium-mediated transformation on FatB3-LPAAT-380DTH (FL), fatB3-KASI-380DTH (FK) and FatB3-LPAAT-KASI-380DTH (FLK), performing electric agrobacterium-mediated transformation on GV3101, performing colony PCR verification, using an agrobacterium dip method to dip wild arabidopsis inflorescence, collecting mature T0 generation seeds through normal growth, screening on a 1/2MS hygromycin (30 mg/ml) plate to obtain hygromycin resistant plants, referring to FIG. 10, performing in-pot growth when T1 generation plants grow to 2-3 leaves on the plate, extracting single plant leaf genomic DNA for PCR positive verification, collecting T1 generation positive plant seeds, performing normal sowing, extracting T2 generation leaf genomic DNA for PCR positive verification, referring to FIG. 11, and showing that three groups of polygenic systems succeed in passage in the electrophoresis diagram, 1 is wild type contrast, 2 and 9 are positive controls, 3-5 generation plants in the FatB3PCR diagram are positive verification, and F3-6-8 is positive verification and F6-12 is positive verification; 3-8 are FLK positive verification and 10-12 are FK positive verification in the KASI PCR electrophoresis chart; in the LPAAT PCR electrophoresis diagram, 3-8 is FLK positive verification, 10-12 is FL positive verification, T2 generation positive plant seeds are normally collected, a hygromycin plate is used for screening the T2 seeds,

if all growth on the plates is normal, it is considered as a homozygous strain. Thus, homozygous lines were obtained as fatB3-LPAAT, fatB3-KASI, and fatB3-LPAAT-KASI, respectively, as the subsequent experimental materials.

Example 4 quantitative analysis of polygenic fluorescence

The promoters of the three groups of polygene systems are seed specific, and total RNA of the T2 generation seeds of the arabidopsis thaliana is extracted for fluorescence quantification to identify the expression condition of polygenes in plants. Fluorescent quantitative PCR data use 2- ^△△ Ct treatment resulted in relative expression differences. FIG. 12 is a fluorescent quantitative result of three genes of fatB3-LPAAT-KASI (FLK), wherein the expression trends of genes among fatB3, LPAAT and KASI lines are similar, and the expression amounts of three genes of line 17 are highest compared by taking line 13 as a standard, and the multiples thereof are 110, 120 and 142 respectively; the fold expression level of the three genes of line 3 was 109, 111, 281; the remaining strains are not much different. FIG. 13 shows the fluorescence quantification results of fatB3-LPAAT (FL), wherein the expression tendencies of two genes of the LPAAT and fatB3 strains are similar, and the comparison is carried out by taking the strain 11 as a standard, wherein the expression amount of the strain 3 is highest, the expression multiples of the fatB3 and the LPAAT are 33 and 4 respectively, the expression multiples of the fatB3 are 23 and 13 respectively, the expression multiples of the other strains are not much different from each other. FIG. 14 shows the fluorescence quantitative results of fatB3-KASI (FK), wherein the expression level of line 9 was highest, the expression fold of fatB3 and KASI was 52 and 13, respectively, and the expression fold of line 12 was 48 and 12, respectively, when compared with line 90; the third highest was 57, the expression fold was 11 and 5, and the remaining strains were not significantly different.

EXAMPLE 5 polygenic Arabidopsis seed fatty acid methyl ester GC assay

The relative content of fatty acids was calculated by GC detection after methyl esterification of T3 generation arabidopsis seed fatty acids extracted according to chloroform: methanol=2:1, and the total fatty acid content was calculated using c17:0 as an internal standard.

Referring to FIG. 15, the relative content trend of FLK fatty acids is substantially consistent, and the C12:0 content is significantly increased compared to the wild type (wild type relative content is 0.035% + -0.00594, FLK minimum relative content is 0.639% + -0.0898), C14:0 relative content was increased (wild type relative content 0.133% ± 0.0147, flk minimum relative content 0.276% ± 0.0333), wherein 17 lines compared to the remaining two lines found C14:0 varies maximally, with a relative content of 1.247% ± 0.0378.

Referring to fig. 16, the relative content trend of fl fatty acid methyl ester was consistent, C12 compared with the wild type: 0 (minimum FL relative content 0.176% ± 0.0136), C14:0 relative content increased (FL minimum relative content 0.644% ± 0.0362), where 34 lines were compared to the remaining two lines, C14: the relative content of 0 varies significantly, with a relative content of 1.96% + -0.130.

Referring to fig. 17, the trend of change in relative content of fk fatty acid methyl ester was consistent, C12 compared with wild type: 0 (minimum relative FK content of 0.079% ± 0.0031), C14:0 relative content increased (FK minimum relative content 0.269% ± 0.0362), where 9 lines were compared to the remaining two lines, C14: the relative content of 0 varies significantly, with a relative content of 1.018% ± 0.0437.

Referring to FIG. 18, a comparison of 17 lines in FLK, 34 lines in FL and 9 lines of FK species revealed that the seed of the fatB3-LPAAT transgenic plant produced C14:0 (relative percentage) 57% and 92% more than the seed of the fatB3-LPAAT-KASI and fatB3-KASI transgenic plants, and that the seed of the fatB3-LPAAT-KASI transgenic plant produced C12:0 (relative percentage) 18% and 157% more than the seed of the fatB3-LPAAT and fatB3-KASI transgenic plants.

Referring to FIG. 19, the fatB3-LPAAT, fatB3-KASI and fatB3-LPAAT-KASI transgenic plant seeds were increased by at least 47%, 70% and 56% respectively over wild type seeds, with no significant difference in total fatty acid content between the three transgenic lines, with 17 and the remaining two of the FLK lines being found to be highest in comparison, with a value of 8.714 + -00.696 μg/50mg, and the wild type Arabidopsis seed content of 4.822 + -0.242 μg/50mg; 34 of FL lines and the other two lines were found to be highest in content by comparison, which was 7.883 + -0.946 μg/50mg, and 12 of FK lines and the other two lines were found to be highest in content by comparison, which was 9.687 + -1.102 μg/50mg.

The principle of the construction of the polygene vector of the invention is as follows:

the Cre/LoxP system is derived from a P1 phage and consists of two parts: a LoxP site capable of recombination, and Cre recombinase capable of recognizing the LoxP site and catalyzing recombination reaction. LoxP site consists of 34bp nucleotide, two ends of the LoxP site contain 13bp inverted repeat sequence, and 8bp asymmetric element is used for determining the direction of LoxP. Cre/LoxP can mediate site-specific insertion, translocation, excision, etc., wherein recombination occurs in three ways: (1) When 2 LoxP loci are positioned on the same molecule and have opposite directions, DNA fragments among the 2 LoxP loci are inverted after recombination; (2) When two LoxP loci are positioned on the same molecule and have the same direction, DNA fragments between 2 LoxP loci are removed after recombination; (3) 2 LoxP sites on different molecules can allow DNA exchange or translocation. The Cre enzyme can recognize not only 13bp inverted repeat and 8bp intermediate sequence of LoxP, but also can recognize and recombine when one 13bp inverted repeat or 8bp intermediate sequence is changed. A multi-gene assembling and transforming carrier system is composed of a receptor carrier (pYLTAC 380 DTH) with LoxP site, two donor carriers (pYL-332-d 1, pYL-332-d 2) with LoxP site and colibacillus NS3529 able to generate Cre recombinase, and features that the target gene (including promoter and terminator) is linked to the donor carrier by molecular biologic means, the donor carrier and receptor carrier are integrated under the action of Cre recombinase to become a big carrier with two LoxP sites, the skeleton of donor carrier is excised to form a new receptor carrier, and the multiple recombination of donor carrier is carried out to form a multi-gene carrier.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> university of Hainan

<120> a method for producing medium-chain fatty acid in plant cell

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1356

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgcaaatct cctttatcac ctataaatta acacatccgc ttcactcttt actcaaacca 60

aaactcatca atacaaacaa gattaaaaac atacacggta cccggggatc catggtcgcc 120

tccgttgctg cctcatcatc tttcttcccg gtcccatctt cctcctcctc ggcctcggca 180

aaagcttcga gaggcatccc agatggtttg gatgtccggg gcatcgtagc gaagccggca 240

tcttcttccg ggtggatgca ggccaaggca agcgcccgag ccatcccaaa aatcgacgac 300

accaaggttg gcctgcggac cgacgtcgag gaggatgccg cttcaacggc gcggagaact 360

tcatataacc aattgccgga ctggagcatg ctgcttgccg cgatcaggac catcttttcg 420

gccgcggaga agcaatggac cctgctcgat tccaagaaga ggggggccga cgcggtcgca 480

gatgcctctg gggtcgggaa gatggtcaag aatggacttg tttacaggca gaatttttct 540

atccggtcct acgaaatcgg ggttgataaa cgtgcttcgg tagaggcatt gatgaatcat 600

ttccaggaaa cgtcgcttaa ccattgcaag tgtattggcc ttatgcatgg cggctttggt 660

tgtacaccag agatgactcg aagaaatctg atatgggttg ttgccaaaat gctggttcat 720

gtcgaacgtt atccttggtg gggagacgtg gttcaaataa atacgtggat tagttcatct 780

ggaaagaatg gtatgggacg tgattggcat gttcatgact gccaaactgg cctacctatt 840

atgaggggta ccagtgtctg ggtcatgatg gataaacaca cgaggagact gtctaaactt 900

cctgaagaag ttagagcaga gataacccct ttcttttcag agcgtgatgc tgttttggac 960

gataacggca gaaaacttcc caagttcgat gatgattctg cagctcatgt tcgaaggggc 1020

ttgactcctc gttggcatga tttcgatgta aatcagcatg tgaacaatgt caaatacgtc 1080

ggctggattc ttgagagcgt tcctgtgtgg atgttggatg gctacgaggt tgcaaccatg 1140

agtctggaat accggaggga gtgtaggatg gatagtgtgg tgcagtctct caccgccgtc 1200

tcttccgacc acgccgacgg ctcccccatc gtgtgccagc atcttctgcg gctcgaggat 1260

gggactgaga ttgtgagggg tcaaacagaa tggaggccta agcagcaggc tcgtgatctt 1320

gggaacatgg gtctgcaccc aactgagagt aaatga 1356

<210> 2

<211> 927

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggatgctt caggggcaag ttcgttcttg cggggccgtt gtctggagag ctgcttcaaa 60

gcgagcttcg ggatgtccca accgaaagat gcagccgggc aaccgagtcg ccggccggcc 120

gacgcggatg actttgtgga tgatgataga tggattactg tcatcctgtc ggtcgttagg 180

atcgctgctt gctttctgtc gatgatggtt accaccatcg tgtggaacat gatcatgctg 240

attttgctcc cttggccata tgctcggatc aggcagggaa acttgtatgg ccatgttacc 300

gggcggatgc tgatgtggat cttagggaac ccaataacaa tagaaggttc tgaattctcg 360

aacacaaggg ccatctacat ctgtaatcat gcatcacttg tagacatttt tctcatcatg 420

tggttgattc caaagggtac cgttaccata gcaaaaaaag agatcatttg gtacccactc 480

tttgggcagc tttatgtatt ggcaaaccat cagcgaatag accggtccaa cccatccgct 540

gccattgagt caattaaaga ggtagctcga gcagttgtca agaaaaactt atcgctgatc 600

atttttccag agggtactcg atcgaaaaca ggaaggctgc ttccatttaa gaagggtttt 660

attcacatag cacttcagac acggttgccg atagttccaa tggtgctgac gggtacccat 720

ctagcttgga ggaagaacag tttgcgagtc agaccagcac ctatcacagt gaaatacttc 780

tcacccataa aaactgatga ctgggaagaa gaaaagatca atcattatgt ggaaatgata 840

catgccttgt acgtggatca cctgccggag tctcaaaaac ctttggtatc aaaagggagg 900

gatgctagcg gaaggtcaaa ttcataa 927

<210> 3

<211> 1485

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggccacaa gtgctagtat cggggtcgcg ggaaaggagt tgggagtgat tagcgaagcc 60

tcatctcctc tcaagcggta taatggccta aggcctttgc tggggagcaa gcagatggct 120

tcctttgctg ctatgaggaa accaaatggt ccctttcctt ctttgccatc tcttaaatct 180

ccaaggataa gaactgtggc ttccccaact gttgctgctc caaagcgaga gaaggatccc 240

aagaaaagga tagtggtgac tgggataggc ctggtctcgg tgtttgggag cgacattgat 300

acattctaca acaagctctt ggaaggacag agcgggatta gcctgatcga ccgatttgat 360

gcttcttctt actctgtccg attcggtgga cagatccggg acttctcctc gaaaggctac 420

attgatggga agaatgatcg ccggctcgat gactgctgga gatattgcct ggtcgccggc 480

aaaagagctc ttgatgatgc taatctcgga ccagaagtcc tgcaatctat ggacaggtcg 540

agaattggag tgctggtagg gacaggcatg ggtggtttaa cggctttcag caatggagtt 600

gaggctctga tccaaaaggg ttacaagaaa attactcctt tcttcattcc ttactccatc 660

acaaacatgg gatcggcgtt gttagctata gaaacaggct taatgggacc aaactactcc 720

atttccacgg catgtgcaac tgcaaactac tgcttttatg ctgctgccaa tcacataagg 780

agaggtgaag ctgacattat ggttgctgga ggaacagagg cggcaatcct gcctactgga 840

gttggtggat tcatcgcatg cagagcactg tcacaaagaa atgatgaacc acagaaagct 900

tcgaggcctt gggacaaaga ccgagatggt ttcgtcatgg gagagggatc tggtgtcctc 960

attatggaga gtctagagca tgcaagaaag aggggtgcaa ctataattgc agagtatctt 1020

ggaggtgcca taacctgtga cgcgcatcac atgaccgatc ctcgttctga tggacttgga 1080

gtctcttctt gcattgttaa gagcttggaa gatgcaggag tctcccccga ggaggtgaat 1140

tatgtcaatg ctcatgcaac atcaacactt gctggagatt tagcagaagt taatgccatc 1200

aagaaggttt tcaaagacac atccgaaatg aaaatgaatg gaacaaagtc gatgattggg 1260

cattgccttg gagctgctgg tgggctggaa gcaattgcaa ccatcaaagc tatcacaaca 1320

ggctggctgc atccaaccat caaccaaaat aacttggagc ctgatgtcac cgtcgacacc 1380

atccccaatg taaagaagaa gcatgaggtt aatgttgcca tctctaattc gtttggtttc 1440

gggggtcaca attctgtggt tgtttttgct cccttcatgc cttaa 1485

<210> 4

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

taggatccat ggtcgcctcc gttgctgcct ca 32

<210> 5

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atgtcgactc atttactctc agttgggtgc a 31

<210> 6

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cactctagat ggatgcttca ggggcaagt 29

<210> 7

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gacctgcagg cggaaggtca aattcataa 29

<210> 8

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atggatccat ggccacaagt gctagtat 28

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atctgcagtt aaggcatgaa gggagcaa 28

<210> 10

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

aagtaagctt ggctacggag tttcgatg 28

<210> 11

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

caaaggtacc acggtttctg ttttatgaaa tg 32

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

aaggtaccta acggtaaaaa aagtagacc 29

<210> 13

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

catggatcct gtttcggagg atctttg 27

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

aagcttctct catccccttt taaac 25

<210> 15

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ggtaccgtgt atgtttttaa tcttgt 26

<210> 16

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tggatgttgg atggctacga ggt 23

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

agaagatgct ggcacacgat gg 22

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

atgctgctgc caatcacata agg 23

<210> 19

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tgcgatgaat ccaccaactc ca 22

<210> 20

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tcaggcaggg aaacttgtat gg 22

<210> 21

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

actcaatggc agcggatgg 19

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gcccctgagg agcacccagt t 21

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ccggttgtac gaccactggc a 21

<210> 24

<211> 9489

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

gaattcgggg gatctggatt ttagtactgg attttggttt taggaattag aaattttatt 60

gatagaagta ttttacaaat acaaatacat actaagggtt tcttatatgc tcaacacatg 120

agcgaaaccc tataggaacc ctaattccct tatctgggaa ctactcacac attattatgg 180

agaaactcga gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgtgtatg 240

tttttaatct tgtttgtatt gatgagtttt ggtttgagta aagagtgaag cggatgtgtt 300

aatttatagg tgataaagga gatttgcatg gcgatcacgt gtaataatgc atgcacgcat 360

gtgattgtat gtgtgtgctg tgagagagaa gctcttaggt gtttgaaggg agtgacaagt 420

gacgaacaaa aacaatcctc cgcgtctgca tgctttgtgt aacgtgtagc taatgttctg 480

gcatggcatc ttatgagcga ttctttttaa aaacaaggta aaaacttaac ttcataaata 540

aaaaaaaacg tttagtaagt tggtttaaaa ggggatgaga gaagcttggc actggccgtc 600

gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca 660

catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa 720

cagttgcgca gcctgaatgg cgaatgctag agcagcttga gcttggatca gattgtcgtt 780

tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga 840

gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt ttatccgttc 900

gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc 960

aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcttctgaaa 1020

acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc 1080

gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac 1140

attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg 1200

acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt 1260

tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc 1320

acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc 1380

gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg 1440

cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg 1500

gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg 1560

ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc 1620

acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg 1680

gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg 1740

aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg 1800

ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga 1860

accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga 1920

gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc 1980

caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa 2040

aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg 2100

atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag 2160

aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc 2220

ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc 2280

gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac 2340

gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac 2400

ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct 2460

tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg 2520

gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc 2580

ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg 2640

cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc 2700

gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt 2760

tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc 2820

cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga 2880

agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac 2940

gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa 3000

tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat 3060

caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg 3120

ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc 3180

gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg 3240

tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc 3300

agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg 3360

gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc 3420

agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga 3480

cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga 3540

gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga 3600

ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga 3660

agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt 3720

ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt 3780

aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac 3840

ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg 3900

gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa 3960

gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg 4020

ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa 4080

gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg 4140

caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc 4200

tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc 4260

ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg 4320

tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa 4380

cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga 4440

tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct 4500

taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa 4560

agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc 4620

ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca 4680

agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt 4740

tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 4800

tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 4860

gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta 4920

tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag 4980

atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct 5040

gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 5100

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 5160

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 5220

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 5280

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 5340

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 5400

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 5460

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 5520

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 5580

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 5640

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5700

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 5760

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 5820

gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact aaaacaattc 5880

atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa 5940

aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc 6000

aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt 6060

gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc 6120

ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc 6180

ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc 6240

ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa 6300

gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata 6360

ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg 6420

ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc 6480

cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata 6540

taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc 6600

ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc 6660

catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat 6720

aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca 6780

gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg 6840

aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc 6900

cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg 6960

gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg 7020

atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc 7080

tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca 7140

cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg atctggattt 7200

tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat tttacaaata 7260

caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct ataggaaccc 7320

taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag cttgtcgatc 7380

gacagatccg gtcggcatct actctatttc tttgccctcg gacgagtgct ggggcgtcgg 7440

tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc gcttctgcgg 7500

gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc gcatcgaccc 7560

tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag ttggtcaaga 7620

ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg atgcctccgc 7680

tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa gaagatgttg 7740

gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac cgctgttatg 7800

cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag gtgccggact 7860

tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac ggacgcactg 7920

acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat cgcgcatatg 7980

aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc 8040

gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg ttgtagaaca 8100

gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg gcgggagatg 8160

caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat cgggagcgcg 8220

gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc gcagctattt 8280

acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga ttcttcgccc 8340

tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa cttctcgaca 8400

gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccgggatc tgcgaaagct 8460

cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct ccaaatgaaa 8520

tgaacttcct tatatagagg aaggtcttgc gaaggatagt gggattgtgc gtcatccctt 8580

acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct 8640

ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca 8700

tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt 8760

tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc 8820

gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg 8880

atattcttgg agtagacgag agtgtcgtgc tccaccatgt tatcacatca atccacttgc 8940

tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca 9000

tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt atcgcaatga 9060

tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg acagatagct 9120

gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt ctcaatagcc 9180

ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc 9240

accatgttgg caagctgctc tagccaatac gcaaaccgcc tctccccgcg cgttggccga 9300

ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg 9360

caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 9420

ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 9480

atgattacg 9489

Claims (1)

1. A method for producing medium-chain fatty acid in plant cells is characterized in that the Cre/LoxP system is adopted to successfully construct the medium-chain fatty acidFatB3-LPAAT、FatB3-KASIOr (b)FatB3-LPAAT-KASIThree polygenic systems and transfer into arabidopsis for seed-specific co-expression, comprising the steps of:

constructing a polygene coexpression vector, preparing agrobacterium competence, transforming the polygene coexpression vector into agrobacterium competence, impregnating arabidopsis thaliana by using an agrobacterium dip method, verifying transgenic arabidopsis thaliana, performing fluorescence quantitative PCR on arabidopsis thaliana seeds, extracting fatty acid of the arabidopsis thaliana seeds, and performing GC determination;

wherein ,FatB3the source of the gene information is GenBank accession: JF338905.1,KASIThe source of the gene information is GenBank accession JX275887,LPAATThe source of the gene information is GenBank accession XP_002522947.1;

the construction of the polygene coexpression vector comprises the following steps:

1) Construction of donor vector:

(1) Respectively byNapin、FatB3And pYL322-d1 orNapin、FatB3And pYL322-d2 plasmid constructionNapin- FatB3-d1/d2 plasmid andNapin-FatB3-the d1/d2 plasmid and pYLTAC380DTH plasmid are mixed, shocked and transferred into the competent NS3529 of the escherichia coli capable of producing Cre recombinase, and after the NS3529 is activated, the mixture is coated on the surface of the plasmid added with Kana ⁺ and Cm⁺ LB solid of (B)Performing expansion culture in a body culture medium, and culturing 22h at 37 ℃;

(2) Extracting plasmid in NS3529 after amplification, performing I-SceI digestion on plasmid, recovering digested fragment, transferring into competent TransT1 of Escherichia coli by heat shock, activating TransT1, and applying to the strain containing Kana ⁺ Culturing 12h on X-gal medium at 37 deg.C, selecting white colony for colony PCR, and screening positive colony;

(3) Amplifying and culturing the screened positive bacterial colony, and extracting plasmid in the amplified and cultured TransT1 bacterial liquid to obtainFatB3-380DTH plasmid;

2) To be used forFatB3、LPAATConstructing a polygene coexpression vector by using a pYLTAC380DTH plasmid;

(1) With a promoter 5400,LPAATAnd pYL322-d1 or by means of the promoter 5400,LPAATAnd construction of pYL-322-d 2 plasmid 5400-LPAATThe d1/d2 plasmid was isolated and isolated as 5400-LPAAT-d1/d2FatB3The 380DTH plasmid was isolated according to 1:1-2:1 in E.coli competent NS3529, after activation of NS3529, it was coated with Kana ⁺ and Amp⁺ Performing expansion culture in LB solid medium, and culturing at 37 ℃ for 22h;

(2) Extracting plasmid in NS3529 after amplification, performing I-SceI digestion on plasmid, recovering digested fragment, transferring into competent TransT1 of Escherichia coli by heat shock, activating TransT1, and applying to the strain containing Kana ⁺ Culturing 12h on X-gal medium at 37 deg.C, selecting white colony for colony PCR, and screening positive colony;

(3) Amplifying and culturing the screened positive bacterial colony, and extracting plasmid in the amplified and cultured TransT1 bacterial liquid to obtainFatB3-LPAAT-380DTH polygene coexpression vector;

3) To be used forFatB3、KASIConstructing a polygene coexpression vector by using a pYLTAC380DTH plasmid: in particular according toFatB3-LPAATConstruction method of-380 DTH polygene coexpression vector16460、KASI and pYL322-d1 or will16460、 KASI and pYL322-d2 construction16460-KASI-d1/d2 plasmid and use16460-KASI-d1/d2 plasmidFatB3-380DTH recombinant constructionFatB3-KASI-380DTH polygene expression vector;

4) To be used forFatB3、LPAAT、KASIConstructing a polygene coexpression vector by using a pYLTAC380DTH plasmid: recombinant construction of a polygene expression vector by 16460-KASI-d1/d2 and FatB3-LPAAT-380 DTH;

the agrobacteria dip-dyeing method for the arabidopsis comprises the following steps:

1) To be used forFatB3-LPAAT、FatB3-KASIOr (b)FatB3-LPAAT-KASIThree polygenic systems are transferred into agrobacterium, and the transformed agrobacterium liquid contains 50ug/mL Rif ⁺ And 50ug/mL Kana ⁺ Streaking on a flat plate of the antibiotic, and culturing for 2-3 days at 28 ℃ in an inverted manner;

2) Picking up single colony for colony PCR, selecting positive colony for inoculation on the surface coated with Rif ⁺ and Kana⁺ In 20mL liquid culture medium of (2), shake culturing at 28 ℃ and 200rpm to OD ₆₀₀ =1.0；

3) Preparing agrobacterium leaching solution according to a proportion, adding 25g of sucrose, 50 mu L of Silwet77 and 0.25g of MES acid and 1.05g of MS powder into the 500mL leaching solution, and supplementing the solution to 500ml by using sterile water;

4) Selecting the period with the most inflorescences of the wild arabidopsis, cutting off the grown fruit clamp, and immersing the inflorescences in the soaking solution for 3min;

5) Placing Arabidopsis thaliana in a basin horizontally after dip dyeing is finished, culturing in the dark for 24h, and repeating dip dyeing after one week; after the dip dyeing is finished, the arabidopsis thaliana is normally placed and cultivated to seed.

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