CN110257444A - A method of producing medium chain fatty acid in plant cell - Google Patents

Abstract

The method that the invention discloses a kind of to produce medium chain fatty acid in plant cell, FatB3-LPAAT is successfully constructed using Cre/LoxP system, tri- kinds of polygenic systems of FatB3-KASI and FatB3-LPAAT-KASI are simultaneously transferred to progress seed specific coexpression in arabidopsis, it include: the building of polygenes coexpression vector, the preparation of Agrobacterium competence, polygenes coexpression vector converts Agrobacterium competence, Agrobacterium dips in colored method dip dyeing arabidopsis, transgenic arabidopsis verifying, arabidopsis seed quantitative fluorescent PCR and arabidopsis Fatty Acids in Seeds extract and carry out the process of GC measurement.The present invention successfully constructs tri- kinds of polygenic systems of FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI using Cre/LoxP system, and it is transferred to progress seed specific coexpression in arabidopsis, three assortment of genes coexpression trend are analyzed, the content for synthesizing medium chain fatty acid in plant cell is effectively increased.

Description

Method for producing medium-chain fatty acid in plant cell

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

The medium chain fatty acid is mainly decomposed by lipase in pancreas and then transported to liver through blood vessel to carry out β oxidation and release energy, the metabolism speed is 10 times of that of long chain fatty acid, and obesity is not easy to form.

The natural medium-chain fatty acid is generally from animal milk products, the rest is mainly extracted from oil palm and coconut, the sources are limited, and the medium-chain fatty acid in the oil palm and the coconut is extracted more complexly and has less extraction amount; the plant genetic engineering can improve the oil content of the plants in a targeted manner so as to improve the oil yield per unit area, and the total yield of the medium-chain fatty acids can be improved by adopting the limited commonly available land and not increasing the planting area. At present, no report is found on a method for producing medium-chain fatty acids by using a genetic engineering means.

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

Disclosure of Invention

In view of the above, the present invention provides a method for producing medium-chain fatty acids in plant cells, which utilizes a genetic engineering means to construct a polygene coexpression vector of FATB3, LPAAT and KASI, and transfers the polygene coexpression vector into Arabidopsis thaliana to produce medium-chain fatty acids in Arabidopsis thaliana, thereby increasing the yield of the medium-chain fatty acids.

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

a method for producing medium-chain fatty acid in plant cells, which adopts Cre/LoxP system to successfully construct three polygene systems of FatB3-LPAAT, FatB3-KASI or FatB3-LPAAT-KASI and transfers the polygene systems into Arabidopsis for seed-specific co-expression, comprises the following steps:

constructing a polygene co-expression vector, preparing agrobacterium infection status, transforming agrobacterium infection status by the polygene co-expression vector, infecting arabidopsis by an agrobacterium dip-flower method, verifying transgenic arabidopsis, performing fluorescence quantitative PCR on arabidopsis seeds, extracting arabidopsis seed fatty acid and performing GC determination.

Alternatively, the construction of the multigene co-expression vector comprises the following steps:

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

Alternatively, the construction of the donor vector comprises:

1) respectively constructing Napin-FatB3-d1/d2 plasmid by Napin, FatB3 and pYL322-d1 or Napin, FatB3 and pYL322-d2 plasmids, transferring the Napin-FatB3-d1/d2 plasmid and pYLTAC380DTH plasmid into escherichia coli competent NS3529 capable of generating Cre recombinase by mixed electric shock, coating the NS3529 in LB solid culture medium added with Kana + and Cm + after the NS3529 is activated, carrying out amplification culture, and culturing for 22h at 37 ℃;

2) extracting the plasmid in the amplified NS3529, carrying out I-SceI enzyme digestion on the plasmid, recovering the fragment after enzyme digestion, carrying out heat shock on the fragment, transferring the fragment into escherichia coli competence TransT1, coating the fragment on an X-gal culture medium added with Kana + after the activation of TransT1, culturing the fragment at 37 ℃ for 12h, selecting a white colony, carrying out colony PCR, and screening out a positive colony;

3) and (3) carrying out amplification culture on the screened positive colonies, and then extracting plasmids in the TransT1 bacterial liquid after the amplification culture to obtain FatB3-380DTH plasmids.

Optionally, the construction method of the multi-gene co-expression vector in step 2) comprises:

1) constructing 5400-LPAAT-d1/d2 plasmid by using promoter 5400, LPAAT and pYL322-d1 or promoter 5400, LPAAT and pYL322-d2 plasmid, and constructing 5400-LPAAT-d1/d2 and FatB3-380DTH plasmid according to the proportion of 1: 1-2: 1, transforming escherichia coli competent NS3529 by electric shock, after NS3529 is activated, coating the activated NS3529 in an LB solid culture medium coated with Kana + and Amp + for propagation, and culturing for 22h at 37 ℃;

2) extracting the plasmid in the amplified NS3529, carrying out I-SceI enzyme digestion on the plasmid, recovering the fragment after enzyme digestion, carrying out heat shock on the fragment, transferring the fragment into escherichia coli competence TransT1, coating the fragment on an X-gal culture medium added with Kana + after the activation of TransT1, culturing the fragment at 37 ℃ for 12h, selecting a white colony, carrying out colony PCR, and screening out a positive colony;

3) and (3) carrying out amplification culture on the screened positive colonies, and then extracting plasmids in the TransT1 bacterial liquid after the amplification culture to obtain the FatB3-LPAAT-380DTH polygene co-expression vector.

Optionally, the multi-gene co-expression vector in the step 3) is constructed by constructing 16460, KASI and pYL322-d1 of promoters or 16460, KASI and pYL322-d2 into 16460-KASI-d1/d2 plasmid according to the construction method of the FatB3-LPAAT-380DTH multi-gene co-expression vector, and recombining the FatB3-KASI-380DTH multi-gene expression vector by using 16460-KASI-d1/d2 plasmid and FatB3-380 DTH.

Optionally, the construction method of the multi-gene co-expression vector in the step 4) is to recombine 16460-KASI-d1/d2 and FatB3-LPAAT-380DTH to construct the FatB3-LPAAT-KASI-380DTH multi-gene expression vector.

Optionally, the method for impregnating arabidopsis thaliana by agrobacterium dipping comprises the following steps:

1) transferring the transformed agrobacterium liquid into agrobacterium by three multigene systems of 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 inversely culturing the streaked agrobacterium liquid at 28 ℃ for 2-3 days;

2) picking up single colony for colony PCR, selecting positive colony, inoculating in 20mL liquid culture medium coated with Rif + and Kana +, shaking culturing at 28 deg.C and 200rpm to OD₆₀₀＝1.0；

3) Preparing an agrobacterium tumefaciens dip dyeing solution according to a ratio, adding 25g of sucrose, 0.25g of Silwet7750 mu L of MES acid and 1.05g of MS powder into 500mL of the dip dyeing solution, and supplementing the mixture to 500mL by using sterile water;

4) selecting the period with the most inflorescences of wild arabidopsis thaliana, cutting off grown fruit clips, and immersing the inflorescences in the dip dyeing solution for 3 min;

5) after the dip dyeing is finished, placing the arabidopsis thaliana in a basin, culturing for 24 hours in the dark, and repeatedly performing dip dyeing after one week; after the completion of the dip dyeing, the Arabidopsis thaliana is normally placed and cultured until seed setting.

Optionally, the kit for PCR positive identification of the DNA of the colony and the transgenic plant is 2xF8 FastLongPCRMasterMix, and the system is as follows:

the amplification system is as follows:

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

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

Optionally, the Primer premier6.0 is used for designing a fluorescence quantitative Primer according to the three gene sequences, the conserved gene β -actin is used as an internal reference Primer, and a fluorescence quantitative kit of Takara is used for real-time fluorescence quantification, wherein the Primer sequences are as follows:

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

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

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

Optionally, when the multigene co-expression vector is constructed, the target fragment and the vector are recovered and purified according to the steps of a GelExtraction Kit (OMEGA).

Optionally, when the multigene co-expression vector is constructed, the PCR system for connecting the target fragment and the vector is as follows:

the PCR cycling conditions were: at 22 deg.C, 60min, 4 deg.C, and infinity.

Optionally, the preparation method of the NS3529 competence comprises:

(1) NS3529 was streaked on non-resistant LB plates at 37 ℃ overnight.

(2) Picking up a single colony, culturing the single colony in 8-10mL of SOB culture medium for 8-12h according to the proportion of 1: inoculating 100 proportion of the seed into 100mL of SOB culture medium, and shake culturing for 2-3h to OD₆₀₀The value is 0.3-0.4.

(3) The mixture was kept on ice for 10min and dispensed into 2 50mL sterile centrifuge tubes and centrifuged at 4000rpm at 4 ℃ for 10 min.

(4) Abandoning the supernatant, adding a small amount of ddH into the centrifuge tube₂O, light suspension precipitation, and then adding ddH₂O was added to 50mL and centrifuged at 4000rpm for 10min at 4 ℃.

(5) Repeating the step (4) once.

(6) Carefully discard the supernatant, add a small amount of pre-cooled sterile 15% glycerol, resuspend the cells, fill up to 50mL, centrifuge at 4 ℃ at 4000rpm for 10min, and discard the supernatant.

(7) Resuspending the thallus with 2-3mL of 15% sterile glycerol, packaging the thallus in 1.5mL sterile EP tube in equal volume of 100uL for use or rapidly freezing with liquid nitrogen and placing in a refrigerator at-80 deg.C for use.

According to the technical scheme, 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, 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 recombinant technology, and then are respectively transferred into wild type Arabidopsis thaliana for seed specific overexpression, and the co-expression synergistic effect of the three genes in the medium-chain fatty acid oil synthesis process is further verified by detecting the expression condition of the genes and the composition and content of fatty acid of transgenic seeds. Wherein, the promoter of FatB3 is a seed specific promoter Napin, and the promoters of LPAAT and KASI are promoters At5400 and At16460 for specific expression in Arabidopsis seed embryo and endosperm respectively; the terminators of all three genes are 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 transgenic arabidopsis T3 generation homozygote fatty acid shows that:

1. FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI transgenic plant seeds produce at least 395%, 124% and 134% increase in C12:0 (relative percentage) over wild type; similarly, C14:0 of the seeds of FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI transgenic plants was also increased by at least 383%, 102% and 106% over wild type seeds; other classes of fatty acids showed no significant differential changes.

2. FatB3-LPAAT transgenic plant seeds yielded 92% and 58% more C14:0 (relative percentage) than FatB3-KASI transgenic plant seeds and FatB3-LPAAT-KASI transgenic plant seeds, respectively, and similarly FatB3-LPAAT-KASI transgenic plant seeds yielded 19% and 116% more C12:0 than FatB3-LPAAT and FatB 3-KASI. The results show that the co-expression of the coconut FatB3 and the LPAAT in the plants has better effect than the co-expression of the three genes FatB-LPAAT-KASI and the two genes FatB3-LPAAT in producing medium-chain fatty acids.

3. The analysis of the total content of medium-chain fatty acids in transgenic seeds shows that the seeds of FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI transgenic plants have at least 47%, 70% and 56% increased respectively compared with wild seeds, and the total content of medium-chain fatty acids in three transgenic lines has no significant difference. The results show that: it is coconut FatB3 and LPAAT that play a major role in coconut medium-chain fatty acid synthesis and accumulation, while 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, the experiment adopts a Cre/LoxP system to successfully construct three polygene systems of FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI, and transfers the polygene systems into Arabidopsis thaliana for seed specificity co-expression, so that the co-expression trend of three gene combinations is analyzed, and the synthetic amount of medium-chain fatty acids in plant cells is increased.

Drawings

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

FIG. 1 is a schematic diagram showing the restriction sites of pYL322-d1 and pYL322-d2 vectors of the present invention;

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

FIG. 3 is a schematic diagram of three target genes and their corresponding promoter sites;

FIG. 4 is a map of pCAMBIA1300S according to the present invention;

FIG. 5 is a flow chart illustrating the construction of a donor vector according to the present invention;

FIG. 6 is a schematic diagram of colony PCR of the promoters Napin, 16460 and the gene KASI according to the present invention;

FIG. 7 is a schematic diagram of colony PCR of the promoter 5400, gene LPAAT and FatB3 of the present invention;

FIG. 8-1 shows the cleavage result of FatB3-380DTH NotI;

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

FIG. 9 is a schematic diagram of single gene location in a multigene vector;

FIG. 10 is a schematic diagram of hygromycin-screened positive plants;

FIG. 11 is a schematic diagram showing PCR positive identification of transgenic Arabidopsis thaliana T2 generation;

FIG. 12 shows the results of fluorescence quantification of the three genes FatB 3-LPAAT-KASI;

FIG. 13 shows the results of fluorescence quantification of FatB3-LPAAT genes;

FIG. 14 shows the results of fluorescence quantification of two genes FatB 3-KASI;

FIG. 15 shows the results of fatty acid methyl ester of FatB3-LPAAT-KASI three genes;

FIG. 16 shows fatty acid methyl esters results of FatB3-LPAAT genes;

FIG. 17 shows fatty acid methyl esters results of FatB3-KASI two genes;

FIG. 18 is a graph showing a comparison of fatty acid results of 17 lines in FLK, FK line 9 and 34 lines in FL;

FIG. 19 shows the total amount of fatty acid methyl esters of the three FatB3-LPAAT-KASI genes and FatB3-LPAAT and FatB3-KASI genes.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example one

Wild type Arabidopsis seeds, Escherichia coli Trans1-T1 competent cells and Agrobacterium tumefaciens GV3101 are all preserved in the laboratory. The Cre/LoxP multi-genome transformation and transformation vector system is provided by Liu dazzling professor laboratory of southern agricultural university, and comprises two vectors pYL322-d1 and pYL322-d2, a receptor vector pYLTAC380DTH and an Escherichia coli strain NS3529 for expressing Cre recombinase, wherein the available enzyme cutting sites of the vectors pYL322-d1 and pYL322-d2 are shown in figure 1, and figure 2 is a schematic diagram of the receptor vector pYLTAC380 DTH.

This example discloses the formation of a donor vector, and in the early stage of the experiment, the inventors have cloned three genes FatB3(GenBank Accession: JF338905.1), KASI (GenBank Accession: JX275887) and LPAAT (GenBank Accession: XP-002522947.1) into pCAMBIA1300S vector.

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 seed maturation process, the promoter 16460 expresses KASI in the seed endosperm formation process, the size is 882bp, the promoter 5400 is started to express LPAAT in the seed endosperm and inner pearl, and the size is 716 bp.

The enzyme cutting sites of the gene and the promoter thereof are designed according to the enzyme cutting sites of the 1300S vector and the donor vector as shown in figure 3, wherein the terminators of the three modules are all from terminator NOS on pCAMBIA 1300S. The map of pCAMBIA1300S is shown in FIG. 4; because the promoter of FatB3 is Napin, firstly, HindIII and KpnI are used for enzyme digestion of 1300S plasmid with Napin promoter, pYL322-d1 plasmid and pYL322-d2 plasmid, target fragment and target vector are recovered, then Napin promoter is cloned to pYL322-d1 vector or pYL322-d2 vector to obtain Napin-d1/d2, colony PCR is used for positive identification, subsequent experiment is carried out after sequencing without mutation, similar method is used, gene FatB3 is cloned to Napin-d1/d2 by using endonuclease BamHI and EcoRI to form donor vector Napin-FatB3-d1/d2, and subsequent experiment is carried out after sequencing without error. The remaining two genes and promoters form an expression module as shown in FIG. 5.

The three expression modules use colony PCR to carry out positive identification, wherein in FIG. 6, electrophoresis channels 3 and 5 are 16460-KASI-d1 promoter 16460 and gene KASI colony PCR amplification results, electrophoresis channels 4 and 6 are 16460-KASI-d2 promoter 16460 and gene KASI colony PCR amplification results, and electrophoresis channels 1 and 2 are Napin-FatB3-d1/d2 promoter Napin colony PCR amplification results respectively; in FIG. 7, electrophoresis channels 3 and 5 are the promoter 5400-LPAAT-d1 (the construction method is the same as that of Napin-FatB3-d1/d 2) and the PCR amplification result of the gene LPAAT colony, and electrophoresis channels 4 and 6 are the promoter 5400 of 5400-LPAAT-d2 and the PCR amplification result of the gene LPAAT colony; the electrophoresis channels 1 and 2 are respectively the colony PCR amplification result of the gene FatB3 of Napin-FatB3-d1/d 2. Through sequencing verification, 6 expression modules are obtained in total, 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).

EXAMPLE 2 construction of the multigenic vector

Mixing Napin-FatB3-d1 plasmid (about 400ng) and pYLTAC380DTH plasmid (about 1 ug) and shocking into E.coli competent NS3529 capable of generating Cre recombinase, after activation, LB plate (Kana +, Cm +) is coated, the culture is carried out for 22h at 37 ℃, sterile water washing plate (the colony number is more than 200) is used for extracting plasmid, I-SceI enzyme digestion is carried out, the recovered heat is transferred into Escherichia coli competence TransT1, activating, coating an X-gal culture medium (Kana +), culturing at 37 ℃ for 12h, selecting white colonies, performing colony PCR, performing amplification culture on positive colonies while verifying the colonies by using an LB plate (Cm +), performing growth verification on chloramphenicol plates without colonies, performing restriction enzyme digestion on plasmids by using Not I, in FIG. 8-2, electrophoresis channel 1 is 380DTH vector NotI enzyme cutting result, electrophoresis channel 2 is FatB3-380DTH vector plasmid NotI enzyme cutting result, and the subsequent implementation is carried out after the sequencing is correct.

In a 5400-LPAAT-d2 plasmid (about 400ng) and a FatB3-380DTH plasmid (about 1. mu.g) as per the proportion, E.coli competent NS3529 was shock-transformed, after activation, LB plate (Kana +, Amp +) is coated, the culture is carried out for 22h at 37 ℃, sterile water washing plate (the colony number is more than 200) is used for extracting plasmid, PI-SceI enzyme digestion is carried out, heat shock is carried out to the Escherichia coli competence TransT1 after recovery, after activation, coating an X-gal culture medium (Kana +), culturing at 37 ℃ for 12h, selecting a white colony for colony PCR, obtaining a positive colony, simultaneously verifying by using an LB plate (Amp +), verifying that the plate has no colony to grow, verifying by using NotI enzyme digestion plasmid, performing subsequent experiments after sequencing is correct, wherein an electrophoresis channel 1 in the graph 8-1 is a 380DTH carrier NotI enzyme digestion result, and an electrophoresis channel 2 is a FatB3-LPAAT-380DTH (FL for short) carrier plasmid NotI enzyme digestion result.

16460-KASI-d2 and FatB3-380DTH obtain a multigene vector FatB3-KASI (FK for short) according to a second-round multigene recombination system, ampicillin LB plates are used for verifying no colony growth and NotI enzyme digestion is used for verifying, an electrophoresis channel 3 in figure 8-1 is FatB3-KASINOTI enzyme digestion result, and subsequent experiments are carried out after sequencing verification.

16460-KASI-d1 and FatB3-LPAAT-380DTH are transformed according to a first wheel set system to obtain a multi-gene vector FatB3-LPAAT-KASI-380DTH, ampicillin LB is used for verifying the growth of a sterile colony, NotI is used for verifying the digestion, an electrophoresis channel 4 in figure 8-1 is FatB3-LPAAT-KASI (FLK for short) NotI digestion result, and subsequent experiments are carried out after sequencing verification.

The relative positions of the single genes in the three multigenic vectors were constructed using DNAMAN, see figure 9, and the multigenic vectors contained hygromycin selection markers, where FatB3 was oriented opposite to LPAAT and KASI in the FL, FK multigenic system, and FatB3 was oriented the same as KASI and opposite to LPAAT, but the orientation of the genes in the multigenic vectors did not affect gene expression.

Example 3 Agrobacterium-mediated Dipigmentation of Arabidopsis thaliana and Positive detection

FatB3-LPAAT-380DTH (FL), FatB3-KASI-380DTH (FK), FatB3-LPAAT-KASI-380DTH (FLK) are electrically transferred to agrobacterium-infected GV3101, wild Arabidopsis inflorescence is dipped by agrobacterium after colony PCR verification is error-free, mature T0 generation seeds are collected in normal growth, hygromycin (30mg/ml) plates are screened to obtain hygromycin resistant plants, see FIG. 10, when T2 generation plants on the plates grow to 387 leaves with 2-3 leaves, the plants are moved to pots in normal conditions for growth, genomic DNA of a single plant leaf is extracted for PCR positive verification, T1 generation positive plant seeds are collected, normal sowing is carried out, genomic DNA of the T2 generation leaf is extracted for PCR positive verification, see FIG. 11, the results show that three groups of successful passage systems are shown, 1 in an electrophoretogram is wild type control, 2. 9 is positive control, 3-5 in a FatB3PCR electrophoretogram is positive verification of FLK, 6-8 is positive verification of FL, and 10-12 is positive verification of FK; 3-8 in KASI PCR electrophoretogram are positive verification of FLK, and 10-12 are positive verification of FK; in the LPAAT PCR electrophoretogram, 3-8 is positive verification of FLK, 10-12 is positive verification of FL, T2 generation positive plant seeds are normally collected, hygromycin plate is used for screening T2 seeds,

homozygous lines were considered if all growth on the plate was normal. The homozygous strains FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI were thus obtained as the subsequent experimental material.

EXAMPLE 4 quantitative analysis of Multi-Gene fluorescence

The promoters of the three groups of multi-gene systems are seed-specific, and the total RNA of seeds of T2 generation of Arabidopsis is extracted for fluorescence quantification to identify the expression condition of the multi-genes in plants. Fluorescent quantitative PCR data use 2-^△△At CtThe relative expression difference is obtained. FIG. 12 shows the results of fluorescence quantification of the three genes of FatB3-LPAAT-KASI (FLK), wherein the gene expression trends among FatB3, LPAAT and KASI strains are similar, and the expression levels of the three genes of strain 17 are the highest by comparison with that of strain 13, and the fold is 110, 120 and 142; the multiple of the expression amount of the three genes of the strain 3 is 109, 111, 281; the other strains are relatively similar. FIG. 13 shows the results of fluorescent quantitation of FatB3-LPAAT (FL), where the expression trends of both genes of the strains LPAAT and FatB3 are similar, and the comparison is performed by using the strain 11 as a standard, where the expression level of the strain 3 is the highest, the expression multiples of FatB3 and LPAAT are 33 and 4, respectively, the expression multiples of the strain 5 and 10 times, and the expression multiples of FatB3 are 23 and 13, respectively, and the differences between the other strains are small. FIG. 14 shows the results of fluorescence quantification of FatB3-KASI (FK), wherein the expression level of strain 9 is highest, the fold expression of FatB3 and KASI is 52 and 13, respectively, and the fold expression of strain is 48 and 12, respectively, when compared with that of strain 90; the third highest is strain 57, whose expression fold is 11, 5, the other strains are comparable.

Example 5 multigenic Arabidopsis seed fatty acid methyl ester GC assay

T3 generation arabidopsis seed fatty acids were extracted according to the method of chloroform-methanol-2: 1, the relative content of fatty acids was calculated using GC assay after methyl esterification, and the total lipid content of fatty acids was calculated using C17:0 as an internal standard.

Referring to fig. 15, the trend of relative content of FLK fatty acids was generally consistent, and the C12:0 content was significantly increased compared to the wild type (relative content of wild type 0.035% ± 0.00594, minimum relative content of FLK 0.639% ± 0.0898), C14: the relative content of 0 is increased (the relative content of a wild type is 0.133% + -0.0147, the minimum relative content of FLK is 0.276% + -0.0333), wherein the comparison of the 17 line and the other two lines shows that C14:0 varied maximally, with a relative content of 1.247% ± 0.0378.

Referring to fig. 16, the relative content of FL fatty acid methyl ester varied consistently, compared to wild type, C12:0 is significantly increased (FL minimum relative content 0.176% ± 0.0136), C14:0 relative content was increased (FL minimum relative content 0.644% ± 0.0362), wherein line 34 was compared with the two remaining lines, C14: the relative content of 0 varies significantly, with a relative content of 1.96% ± 0.130.

Referring to fig. 17, the relative content of FK fatty acid methyl ester varied consistently, compared to the wild type, C12:0 is significantly increased (the lowest relative content of FK is 0.079% ± 0.0031), C14:0 relative content was increased (FK minimum relative content 0.269% ± 0.0362), wherein line 9 was compared to the two remaining 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 lines 17 in FLK, 34 in FL and 9 in FK species revealed that seeds of the FatB3-LPAAT transgenic plants produced 57% and 92% more C14:0 (relative percentage) than seeds of the FatB3-LPAAT-KASI and FatB3-KASI transgenic plants, and seeds of the FatB3-LPAAT-KASI transgenic plants produced 57: 0 (relative percentage) than seeds of the FatB3-LPAAT and FatB3-KASI transgenic plants produced 18% and 157% more C14: 0.

Referring to FIG. 19, in terms of total fatty acid content of Arabidopsis seeds, the seeds of FatB3-LPAAT, FatB3-KASI and FatB3-LPAAT-KASI transgenic plants were increased by at least 47%, 70% and 56% respectively compared with wild-type seeds, and there was no significant difference in total fatty acid content among the three transgenic lines, where 17 in the FLK line was found to be the highest content compared with the two remaining lines, with a value of 8.714 + -00.696 μ g/50mg and the wild-type Arabidopsis seed content was 4.822 + -0.242 μ g/50 mg; the highest content was found in comparison of 34 in the FL strain and the two remaining strains, which was 7.883 + -0.946. mu.g/50 mg, and in comparison of 12 in the FK strain and the two remaining strains, which was 9.687 + -1.102. mu.g/50 mg.

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

the Cre/LoxP system is derived from the P1 bacteriophage and consists of two parts: LoxP sites capable of recombination, Cre recombinase capable of recognizing LoxP sites and catalyzing recombination reactions. The LoxP site consists of 34bp nucleotides, each with a13 bp inverted repeat at both ends and an 8bp asymmetric element in the middle to determine the orientation of LoxP. Cre/LoxP can mediate site-specific insertion, translocation, excision and the like, wherein the recombination has the following three modes: (1) when the 2 LoxP sites are located in the same molecule and the directions are opposite, the DNA fragments among the 2 LoxP sites are inverted after recombination; (2) two LoxP sites are positioned in the same molecule and have the same direction, and DNA fragments between 2 LoxP sites are removed after recombination; (3) the 2 LoxP sites are on different molecules, allowing DNA exchange or translocation. The Cre enzyme recognizes not only 13bp inverted repeats and 8bp intermediate sequences of LoxP, but also a13 bp inverted repeat or an 8bp intermediate sequence when they are changed, and can recognize and recombine. A multi-gene assembling and transforming carrier system includes a receptor carrier (pYLTAT 380DTH) with LoxP sites, two donor carriers (pYL332-d1, pYL332-d2) with LoxP sites individually and colibacillus NS3529 capable of generating Cre recombinase, firstly, the target gene (containing promoter and terminator) is connected to the donor carrier by molecular biological means, the donor carrier and the receptor carrier are integrated into a large carrier with two LoxP sites under the action of Cre recombinase, the donor carrier skeleton is excised after being identified by Cre recombinase to form a new receptor carrier, the latter donor carrier is recombined for many times by alternation with the large carrier to form a multi-gene carrier to construct, the system has simple and efficient advantages, and can contain up to more than ten genes for common expression.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

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 acids in plant cells

<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 (7)

1. A method for producing medium-chain fatty acid in plant cells is characterized in that a Cre/LoxP system is adopted to successfully construct three polygene systems of FatB3-LPAAT, FatB3-KASI or FatB3-LPAAT-KASI and is transferred into Arabidopsis thaliana for seed-specific co-expression, and the method comprises the following steps:

constructing a polygene co-expression vector, preparing agrobacterium infection status, transforming agrobacterium infection status by the polygene co-expression vector, infecting arabidopsis by an agrobacterium dip-flower method, verifying transgenic arabidopsis, performing fluorescence quantitative PCR on arabidopsis seeds, extracting arabidopsis seed fatty acid and performing GC determination.

2. The method of claim 1, wherein the construction of the multiple gene co-expression vector comprises the steps of:

1) constructing a donor vector; 2) constructing a polygene co-expression vector by FatB3, LPAAT and pYLTAC380DTH plasmids; 3) constructing a polygene co-expression vector by FatB3, KASI and pYLTAC380DTH plasmids; 4) the multiple gene co-expression vector was constructed with FatB3, LPAAT, KASI, and pYLTAC380DTH plasmids.

3. The method of claim 2, wherein the donor vector is constructed by:

1) respectively constructing Napin-FatB3-d1/d2 plasmid by Napin, FatB3 and pYL322-d1 or Napin, FatB3 and pYL322-d2 plasmids, shocking the mixture of Napin-FatB3-d1/d2 plasmid and pYLTAC380DTH plasmid into E.coli competent NS3529 capable of generating Cre recombinase, and after NS3529 is activated, coating the E.coli competent NS3529 with Kana⁺ and Cm⁺The LB solid culture medium is subjected to expanding culture and cultured for 22 hours at 37 ℃;

2) extracting plasmid from NS3529, performing I-SceI digestion, recovering the digested fragment, heat-shocking into Escherichia coli competent TransT1, activating TransT1, and coating with Kana⁺Culturing the bacterial strain in the X-gal culture medium at 37 ℃ for 12 hours, selecting a white bacterial colony, carrying out colony PCR, and screening out a positive bacterial colony;

3) and (3) carrying out amplification culture on the screened positive colonies, and then extracting the plasmid in the TransT1 bacterial liquid after the amplification culture to obtain a FatB3-380DTH plasmid.

4. The method of claim 2, wherein the multi-gene co-expression vector of step 2) is constructed by:

1) with promoter 5400, LPAAT andpYL322-d1 or 5400-LPAAT-d1/d2 plasmid constructed by promoter 5400, LPAAT and pYL322-d2 plasmids, and 5400-LPAAT-d1/d2 and FatB3-380DTH plasmids are expressed according to the following steps of 1: 1-2: 1 in a proportion of the total amount of the electric shock transformed E.coli competent NS3529, which was smeared with Kana after activation of NS3529⁺ and Amp⁺The LB solid culture medium is subjected to expanding culture and cultured for 22 hours at 37 ℃;

2) extracting plasmid from NS3529, performing I-SceI digestion, recovering the digested fragment, heat-shocking into Escherichia coli competent TransT1, activating TransT1, and coating with Kana⁺Culturing the bacterial strain in the X-gal culture medium at 37 ℃ for 12 hours, selecting a white bacterial colony, carrying out colony PCR, and screening out a positive bacterial colony;

3) and (3) carrying out amplification culture on the screened positive colonies, and then extracting plasmids in the TransT1 bacterial liquid after the amplification culture to obtain the FatB3-LPAAT-380DTH polygene co-expression vector.

5. The method for producing medium-chain fatty acids in plant cells as claimed in claim 2, wherein the multi-gene co-expression vector in step 3) is constructed by constructing a multi-gene co-expression vector of FatB3-LPAAT-380DTH by constructing 16460, KASI and pYL322-d1 or 16460, KASI and pYL322-d2 into a 16460-KASI-d1/d2 plasmid, and by recombining the 16460-KASI-d1/d2 plasmid and FatB3-380DTH to construct a multi-gene expression vector of FatB3-KASI-380 DTH.

6. The method of claim 2, wherein the multiple gene co-expression vector of step 4) is constructed by recombining 16460-KASI-d1/d2 and FatB3-LPAAT-380DTH to construct a multiple gene expression vector.

7. The method of claim 6, wherein the Agrobacterium dip-dyeing of Arabidopsis thaliana comprises the steps of:

1) with FatB3-LPAAT, FatB3-KASI or FatB3-LPAAT-KASI three polygene systems are transferred into agrobacterium, and the transformed agrobacterium liquid contains 50ug/mL Rif⁺And 50ug/mL Kana⁺Streaking antibiotic plate, and culturing at 28 deg.C for 2-3 days;

2) picking up single colony for colony PCR, selecting positive colony and inoculating to Rif-coated colony⁺ and Kana⁺20mL of the culture medium was cultured at 28 ℃ with shaking at 200rpm until OD₆₀₀＝1.0；

3) Preparing an agrobacterium tumefaciens dip dyeing solution according to a ratio, adding 25g of sucrose, 0.25g of Silwet7750 mu L of MES acid and 1.05g of MS powder into 500mL of the dip dyeing solution, and supplementing the mixture to 500mL by using sterile water;

4) selecting the period with the most inflorescences of wild arabidopsis thaliana, cutting off grown fruit clips, and immersing the inflorescences in the dip dyeing solution for 3 min;

5) after the dip dyeing is finished, placing the arabidopsis thaliana in a basin, culturing for 24 hours in the dark, and repeatedly performing dip dyeing after one week; after the completion of the dip dyeing, the Arabidopsis thaliana is normally placed and cultured until seed setting.