CN118064497A - PfbZIP52 gene over-expression vector, construction method and application in tobacco - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a PfbZIP gene over-expression vector, a construction method and application thereof in tobacco. The nucleotide sequence of PfbZIP gene is shown in SEQ ID NO.3, the over-expression vector is obtained by connecting PfbZIP gene with a plant expression vector, the plant expression vector comprises XbaI and KpnI enzyme cutting sites, the PfbZIP gene is connected between the XbaI and KpnI enzyme cutting sites, and the over-expression vector can be used for PfbZIP gene function research and can improve the tobacco fat content by regulating fatty acid metabolism. The PfbZIP gene provided by the invention can improve the content of the tobacco oil and the quality of the oil by regulating and controlling the fatty acid metabolism, and can be used for variety cultivation of tobacco.

Description

PfbZIP52 gene over-expression vector, construction method and application in tobacco

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a PfbZIP gene over-expression vector, a construction method and application thereof in tobacco.

Background

Perilla (Perillafrutescens (l.) britt.) is an annual herb of the genus Perilla of the family Labiatae. The perilla seeds are rich in grease, the oil yield is up to 46% -58%, and the perilla seeds contain rich unsaturated fatty acid, especially alpha-linolenic acid (alpha-LinolenicAcid, ALA). The research shows that the alpha-linolenic acid has high protection effect on the brain nerve function and retina function of human body, can soften blood vessel, promote blood circulation, prevent cardiovascular diseases and coronary heart disease, and is also helpful for improving memory, delaying aging, etc.

Plant fatty acid metabolism is an important biological process in the growth and development process of plants, and relates to multiple aspects of energy storage, cell membrane composition, signal transduction and the like. In recent years, with the rapid development of molecular biology and gene editing techniques, attempts have been made to improve plant growth and quality by regulating genes associated with fatty acid metabolism.

Several studies have shown that plant growth, abiotic stress response, nutrient metabolism, etc. are all affected by transcription factors such as MYB, WRI1, WRKY, bZIP, NAC, etc. Among them, bZIP transcription factors are a family of transcription factors which are widely present in plants and have a large number of kinds. However, the regulation factor for regulating the high-level accumulation of unsaturated fatty acid in perilla plant seeds and the detailed function thereof are not clear at present, so that researches for constructing related vectors at a molecular level for regulating plant growth are rarely carried out, and the application of the regulation factor in transgenic plants is limited.

Tobacco is an annual herb plant of the genus Nicotiana of the family Solanaceae, and is also an important cash crop, the leaves of the tobacco can be used for manufacturing tobacco products such as cigarettes, cigars, pipes and the like, the internal fragrance and the appearance oil content of the tobacco mainly come from contained grease, and the higher the grease content of the tobacco, the better the quality. However, the existing tobacco has the problem of poor quality of grease in the cultivation process.

Therefore, how to improve the content of the tobacco oil and fat by regulating and controlling the fatty acid metabolism in the tobacco cultivation process and improve the quality of the oil and fat are the problems to be solved urgently at present.

Disclosure of Invention

In order to improve the content of the oil in the tobacco, the invention provides an over-expression vector of a transcription factor PfbZIP gene related to the metabolism of plant fatty acid, a construction method and application thereof in the tobacco, and the PfbZIP gene provided by the invention can improve the total content of the oil in tobacco plants.

The invention provides an over-expression vector of PfbZIP genes, the nucleotide sequence of the PfbZIP genes is shown as SEQ ID NO.3, the over-expression vector is obtained by connecting PfbZIP genes with a plant expression vector, the plant expression vector comprises Xba I and Kpn I enzyme cutting sites, the PfbZIP genes are connected between the Xba I and Kpn I enzyme cutting sites, and the over-expression vector can be used for PfbZIP gene function research and can improve the content of tobacco oil and fat by regulating fatty acid metabolism.

Further, the plant expression vector is any one of pCAMBIA1303 vector, pCAMBIA1391Z vector and pCAMBIA1302 vector.

The invention also provides a construction method of the PfbZIP gene over-expression vector, which comprises the following steps:

Amplifying a target gene PfbZIP shown as SEQ ID NO.3 by PCR;

Recovering and purifying the target gene PfbZIP to obtain a target gene PfbZIP52 fragment;

The pCAMBIA1303 vector plasmid is subjected to double-enzyme tangential linearization, purification and recovery to obtain a pCAMBIA1303 vector linear fragment;

connecting the recovered target gene PfbZIP fragment with the pCAMBIA1303 vector linearization fragment to obtain a recombinant product pCAMBIA 1303-PfbZIP;

the recombinant product pCAMBIA1303-PfbZIP is transformed by DH5 alpha competent cells to obtain the overexpression vector of PfbZIP gene.

Further, the reaction system of PCR amplification is as follows: FastPfu FlyPCR SuperMix. Mu.L, 1. Mu.L of cDNA template, 1. Mu.L of each primer pair PfbZIP-1303 (10. Mu.M), and no nuclease water up to 50. Mu.L.

Further, the system for obtaining the recombinant product pCAMBIA1303-PfbZIP by connection is as follows: the target gene PfbZIP fragment 2 mu L, pCAMBIA1303 vector linearized fragment 3 mu L, 2X ClonExpress Mix mu L.

The invention also provides application of the PfbZIP gene over-expression vector in improving the quality of tobacco grease.

Further, the nucleotide sequence of the PfbZIP gene is shown as SEQ ID NO.3, the over-expression vector is obtained by connecting the PfbZIP gene with a plant expression vector, the plant expression vector comprises Xba I and Kpn I enzyme cutting sites, the PfbZIP gene is connected between the Xba I and Kpn I enzyme cutting sites, the over-expression vector is transferred into tobacco to construct transgenic tobacco, and the content of tobacco oil is improved by regulating fatty acid metabolism.

Further, the construction steps of the transgenic tobacco are as follows:

Constructing PfbZIP gene over-expression vector;

Transforming the PfbZIP gene over-expression vector into agrobacterium GV3101 to obtain agrobacterium GV3101 bacterial liquid;

culturing tobacco leaves on a tobacco genetic transformation preculture medium;

Infecting the cultured tobacco leaves with agrobacterium GV3101 bacterial liquid to obtain transgenic tobacco leaves;

transferring the transgenic tobacco leaves to a screening culture medium for culture until cluster buds grow out;

transferring the cluster buds into a rooting culture medium for culture, and obtaining positive transgenic tobacco plants after the transgenic tobacco leaves grow out of root systems.

Further, the pre-culture medium formula is 4.74g MS culture medium powder, 30g/L sucrose, 7g/L agar, 1.0 mg/L6-BA and 0.1mg/LNAA;

the co-culture medium formula is 4.74g MS culture medium powder, 30g/L sucrose, 7g/L agar, 1.0 mg/L6-BA and 0.1mg/LNAA.

Further, the screening culture medium formula is 4.74g MS culture medium powder, 30g/L sucrose, 7g/L agar, 1.0 mg/L6-BA, 0.1mg/LNAA, 500mg/LCef and 5mg/LHyg;

the rooting culture medium is 2.37g of MS culture medium powder, 30g/L of sucrose, 7g/L of agar and 500mg/LCef.

Compared with the prior art, the invention has the beneficial effects that:

(1) The PfbZIP gene provided by the invention can improve the content of the tobacco oil and the quality of the oil by regulating and controlling the fatty acid metabolism, and can be used for variety cultivation of tobacco.

(2) The PfbZIP gene provided by the invention can promote the accumulation of the oil synthesis of perilla oil crops, proves that the PfbZIP gene is related to the regulation and control of fatty acid enrichment, and plays an important role in the synthesis process of the oil TAG stored in plant seeds.

(3) The PfbZIP gene provided by the invention performs space-time expression analysis on different tissues and different seed development periods of perilla, and genetic transformation tobacco function analysis, thereby providing a theoretical basis for discussing the regulation mechanism of PfbZIP gene and gene utilization.

(4) The kana resistance genes of the pCAMBIA1303 vector, the pCAMBIA1391Z vector and the pCAMBIA1302 vector enable the vector to be conveniently screened in escherichia coli, and meanwhile, the kana resistance genes have hygromycin screening marker genes, so that the kana resistance genes can be conveniently screened and identified in plant transformant lines.

Drawings

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

FIG. 1PfbZIP shows the electrophoretogram of PCR amplification products of the gene;

In the figure, M: DL2000 marker,1: pfbZIP52 gene amplification products.

FIG. 2 shows a double digestion electrophoresis of pCAMBIA1303-PfbZIP recombinants;

In the figure, M: DL15000 marker,1: pCAMBIA1303 vector plasmid, 2: the pCAMBIA1303-PfbZIP recombinant was digested with two enzymes.

FIG. 3 shows basic sequence characteristics of the encoding protein of the perilla PfbZIP gene;

In the figure, a: pfbZIP52 gene encodes the functional domain of a protein;

B: pfbZIP52 gene structure;

c: the PfbZIP gene encodes a protein that retains the Motif.

FIG. 4 analysis of the time-space expression characteristics of the Perilla PfbZIP gene.

FIG. 5 is an electrophoretogram of transgenic tobacco positive plant detection;

in the figure, a: detecting an electrophoresis chart by using a transgenic tobacco genome level PCR;

b: detecting an electrophoresis chart by PCR of the transcription level of transgenic tobacco;

m: DL2000 marker,1: pCAMBIA1303-PfbZIP control plasmid, 2: wild type tobacco (WT) plants, 3-7: transgenic (OE) tobacco plants amplified to lengths 1161bp (A) and 250bp (B).

FIG. 6 is a graph of the results of determination of total lipid and fatty acid component content of transgenic tobacco;

in the figure, a: total oil content of transgenic tobacco;

b: the main fatty acid component and the content of the transgenic tobacco;

WT: wild type tobacco plants, OE-1/OE-2/OE-3: transgenic tobacco plants.

FIG. 7 is a graph of the results of other agronomic trait testing of transgenic tobacco;

In the figure, a: transgenic tobacco soluble sugar content;

b: total protein content of transgenic tobacco;

c: starch content of transgenic tobacco;

D: germination rate of transgenic tobacco seeds;

E: thousand seed weight of transgenic tobacco seeds;

F: transgenic tobacco leaf photosynthetic rate.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1: pfbZIP52 gene and construction of overexpression vector thereof

1. Experimental materials

The E.coli strain DH5 alpha, the Agrobacterium tumefaciens strain GV3101 and the pCAMBIA1303 vectors are provided by the institute of molecular agriculture and bioenergy at Shanxi university. All primers are prepared by the technology of the Optimago (western) stock, inc.

The perilla material is a preferred variety 'Jinsu NO. 1', and is provided by molecular agriculture and bioenergy research institute of Shanxi university.

The reagents used were high-fidelity Mix (AS 231, beijing full-size gold biotechnology Co., ltd.), agar gel DNA recovery kit (new scene biological reagent development Co., hangzhou), plasmid extraction kit (new scene biological reagent development Co., ltd.), restriction endonuclease reagent (beijing full-size gold biotechnology Co., ltd.), PCR product purification recovery kit (EP 101, beijing full-size gold biotechnology Co., ltd.), one-step cloning kit (C115, nanjinopran biotechnology Co., ltd.), LB broth, LB nutrient agar, kanamycin (Kan) (beijing solybao technologies Co., ltd.), 2×E-Taq PCR MasterMix (new scene biological reagent development Co., hangzhou). All primers are prepared by the technology of the Optimago (western) stock, inc.

2. Obtaining of perilla PfbZIP gene and construction of its over expression carrier

1. Extraction of Total RNA

Total RNA from each tissue of Perilla frutescens was extracted using EASYspin plant RNA quick extraction kit (Beijing Edley Biotechnology Co., ltd.). Fresh samples of perilla roots, stems, leaves, flowers and seeds of different developmental stages (10 d, 20d, 30d and 40d after flowering, species a, b, c and d) were quick frozen with liquid nitrogen and rapidly ground into powder with a mortar. Adding 750 mu L of lysate into a 1.5mL nuclease-free centrifuge tube, adding 75 mu L PLANTAID, and uniformly mixing for later use; adding 50mg-100mg of each ground sample into powder, and immediately and vigorously shaking and uniformly mixing for 20s to enable the samples to be fully cracked; centrifuging at 13000rpm at 4deg.C for 10min, transferring the supernatant to a new 1.5mL RNase free centrifuge tube, adding half of absolute ethanol, and immediately blowing and sucking; sucking the mixture into an adsorption column RA (the adsorption column is placed in a collecting pipe), centrifuging at 13000rpm for 2min, and discarding the waste liquid; adding 700 mu L deproteinized liquid RW1 into an adsorption column, standing for 1min at room temperature, centrifuging for 30s, and discarding the waste liquid; adding 500 μl of rinsing solution RW (absolute ethanol is added before use), centrifuging at 13000rpm for 30s, discarding the waste liquid, and repeating the steps once; placing the adsorption column RA into a collecting pipe, centrifuging at 13000rpm for 2min; placing the adsorption column into a new nuclease-free centrifuge tube, adding 30-50 μl of nuclease-free water (RNASE FREE WATER) into the middle part of the adsorption membrane (heating in 70-90deg.C metal bath before use), standing at room temperature for 1min,12000rpm, centrifuging for 1min to obtain total RNA of each tissue of Perilla frutescens, and using in subsequent experiments or in-80deg.C ultralow temperature refrigerator for use.

2. First Strand Synthesis of cDNA

The first strand of cDNA was synthesized by reverse transcription using STARSCRIPT II genome-free DNA reverse transcription premixing reagent (Beijing Kang Runcheng Biotechnology Co., ltd.) using the extracted total RNA of each tissue of Perilla as a template.

3. Designing specific primers

Screening and identifying full-length coding sequence (CDS) of PfbZIP gene (C2S51_ 035357) from Perilla genome and transcriptome database;

primers were designed with the pCAMBIA1303 vector homology arm: primer pairs consisting of nucleotide sequences AACCTGCAGGTCGACTCTAGAATGGATGGAGGTTGTGAGCAA (SEQ ID NO. 1) and GGTTTAAACGAGCTCGGTACCTCAGTAAGGACAGCTGAAACTCCTC (SEQ ID NO. 2), the primer pairs being designated PfbZIP-1303.

4. PCR amplification

Performing high-fidelity PCR amplification by taking cDNA mixed samples of all tissues of the perilla obtained after reverse transcription as templates to obtain PCR amplification products;

PCR reaction 50. Mu.L: FastPfu Fly PCR SuperMix. Mu.L, 1. Mu.L of cDNA template, 1. Mu.L of each of SEQ ID NO.1 and SEQ ID NO.2 (10. Mu.M), and Nuclease-free water (nucleic-FREE WATER) were made up to 50. Mu.L;

PCR reaction procedure: 98 ℃ for 1min;98℃for 10s,60℃for 5s,72℃for 10s,35 cycles; 72 ℃ for 1min. The PCR amplified products were stored at 4℃or subjected to agarose gel electrophoresis.

Agarose gel electrophoresis (FIG. 1) combined with sequencing results show that PfbZIP gene has a clear band at 1161bp (nucleotide sequence is shown as SEQ ID NO. 3).

5. PCR amplification product recovery and purification

And (3) using a DNA purification recovery kit to recover a target band of the PCR amplified product detected by agarose gel electrophoresis to obtain a target gene PfbZIP fragment, and preserving at-20 ℃ for later use. The operation steps are as follows: agarose gel containing the DNA fragment of the target gene is cut off under an ultraviolet lamp and transferred into a 1.5mL centrifuge tube; adding 500 mu L Buffer G into the centrifuge tube, and carrying out water bath at 50 ℃ until the gel is completely dissolved; 200 mu L of isopropanol is added and mixed uniformly; transferring the sol solution into a nucleic acid purification column (the nucleic acid purification column is placed in a 2mL centrifuge tube), centrifuging at 12000rpm for 30s, and discarding the waste liquid; adding 500 mu LBufferWS to a nucleic acid purification column, centrifuging at 12000rpm for 30s, and discarding the waste liquid; adding 700 mu LBufferWG, centrifuging at 12000rpm for 30s, discarding the waste liquid, and repeating the steps once; the nucleic acid purification column was placed in a clean 1.5mL centrifuge tube (provided by kit) after centrifugation at 14000rpm for 1min, 25-30 mu LBufferTE was added to the center of the membrane of the purification column, and the DNA was eluted by standing at room temperature for 1min and centrifugation at 12000rpm for 30s to obtain the target gene PfbZIP fragment.

6. Double enzyme cutting to obtain pCAMBIA1303 vector linear fragment

Transforming the pCAMBIA1303 vector with an escherichia coli DH5 alpha competent cell to obtain an escherichia coli DH5 alpha strain containing the pCAMBIA1303 vector, and preserving at-80 ℃; then taking out the escherichia coli DH5 alpha strain containing the pCAMBIA1303 vector from the temperature of minus 80 ℃, immediately thawing on ice, inoculating into LB liquid medium containing kanamycin, and carrying out shaking culture in a constant temperature shaking table at 37 ℃ to obtain escherichia coli bacterial liquid containing the pCAMBIA1303 empty vector for later use;

extraction of pCAMBIA1303 vector plasmid: the pCAMBIA1303 vector plasmid was extracted using Hangzhou New scenery reagent development Limited plasmid extraction kit, as follows: 3mL of the cultured E.coli bacterial liquid containing pCAMBIA1303 empty vector is collected by centrifugation at 12000rpm for 30s, and the supernatant is discarded; adding 250 μl Buffer I (RNaseA before use), and re-suspending thallus precipitate; add 250 μ LBuffer II, gently and fully invert the centrifuge tube 5 times; 350 mu LBufferN of added, gently and fully turning the centrifuge tube until the residual blue precipitate in the solution is completely converted into yellowish precipitate; centrifuging at 12000rpm for 2min; placing the nucleic acid purification column in a 2mL centrifuge tube, pouring the supernatant into the nucleic acid purification column, centrifuging at 12000rpm for 30s, and discarding the waste liquid; adding 800 mu LBufferW < 2 >, centrifuging at 12000rpm for 30s in a nucleic acid purification column, and discarding the waste liquid; the nucleic acid purification column was placed back into a 2mL centrifuge tube and centrifuged at 12000rpm for 1min; placing the nucleic acid purification column in a clean 1.5mL centrifuge tube, adding 60 mu LBuffer E at the center of the membrane of the purification column, standing at room temperature for 1min, and centrifuging at 12000rpm for 30s; the purification column was discarded, and the pCAMBIA1303 vector plasmid was collected into a centrifuge tube and stored at-20℃for further use.

The results of the detection by agarose gel electrophoresis showed that the pCAMBIA1303 vector plasmid had a clear band at 12kb (1 of FIG. 2).

Vector plasmid double enzyme tangentially-cutting of pCAMBIA 1303: the double enzyme digestion reaction system is as follows: pCAMBIA1303 vector plasmid 2. Mu.g, 10U/. Mu.L Xba I1. Mu.L, 10U/. Mu.L Kpn I1. Mu.L,Buffer 5. Mu.L, nuclease-free water (nucleic-FREE WATER) up to 50. Mu.L; the procedure for the double cleavage reaction was as follows: the enzyme digestion is carried out for 2 hours at 37 ℃ and the inactivation is carried out for 20 minutes at 80 ℃.

Purifying and recovering the pCAMBIA1303 vector plasmid linear fragment: the linear fragment of the pCAMBIA1303 vector after double enzyme digestion is purified and recovered by using Quan Shi gold PCR product purification recovery kit, and the method is as follows: taking 50 mu LPCR product, adding 5 times volume of solution BB, mixing, adding into a centrifugal column (standing for 1min for improving purification yield), centrifuging for 1min at 10000g, and discarding the waste liquid; adding 650 mu L of solution WB, centrifuging 10000g for 1min, and discarding the waste liquid; centrifuging at 10000g for 1-2min to thoroughly remove residual WB; the centrifugation column was placed in a clean centrifuge tube, 30-50. Mu.L of EB was added in the center of the column (to increase purification yield, pre-heated EB at 65℃was selected, left standing at room temperature for 1min,10000g was centrifuged for 1min, and DNA was eluted to obtain pCAMBIA1303 vector linear fragment, which was stored at-20℃for use.

7. Obtaining the ligation product pCAMBIA1303-PfbZIP52

According to ClonExpress Ultra One Step Cloning Kit kit (Nanjinouzan biotechnology Co., ltd.) instruction method, the recovered target gene PfbZIP fragment and pCAMBIA1303 vector linearization fragment are connected to obtain recombinant product pCAMBIA1303-PfbZIP52, and stored for standby;

10. Mu.L of the recombinant system was as follows: 3 mu L of target gene PfbZIP fragment 2 mu L, pCAMBIA1303 vector linearization fragment 2X ClonExpress Mix mu L; the recombination reaction is as follows: the reaction was carried out at 50℃for 5min.

8. Plasmid transformation

Taking out competent cells of Escherichia coli DH5 alpha from a refrigerator at-80 ℃, immediately thawing on ice, adding 8 mu L of recombinant product pCAMBIA1303-PfbZIP into 100 mu L of DH5 alpha competent cells, uniformly mixing the walls of a flick centrifuge tube, and carrying out ice bath for 30min, wherein the addition amount of a connection product is not more than one tenth of the volume of the competent cells; placing the centrifuge tube in a metal bath at 42 ℃ for heat shock for 90s, and then immediately placing in an ice bath for 3min; 900 mu L of LB liquid medium without antibiotics is added into the centrifuge tube, and the culture is carried out for 1h at 37 ℃ under 200rpm shaking; centrifuging at 5000rpm for 5min, discarding 900 μl of supernatant, re-suspending thallus with the rest LB medium, and uniformly coating the bacterial liquid on the preheated LB plate medium containing Kan at 37deg.C with a coater; placing into a constant temperature incubator at 37 ℃, and culturing for about 12 hours in an inverted manner; selecting white single colony in 1mL LB liquid medium containing 50mg/mL Kan, sealing with sealing film, shaking at 37deg.C and 200rpm overnight, and culturing to obtain bacterial liquid containing pCAMBIA1303-PfbZIP 52;

9. Recombinant identification

1. Mu.L of the obtained bacterial liquid containing pCAMBIA1303-PfbZIP is taken as a template for PCR amplification to obtain recombinant plasmids. The size of the amplified product fragment was detected by 1% agarose gel electrophoresis, and simultaneously, the recombinant plasmid was subjected to double digestion identification by Xba I and Kpn I.

The PCR amplification system was 10. Mu.L as follows: PCR template 0.5. Mu.L, SEQ ID NO.1 and SEQ ID NO.2 primers each 0.2. Mu.L, 2 XE-Taq PCR MasterMix. Mu. L, ddH ₂ O4.1. Mu.L;

The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 80s,30 cycles; finally, the extension is carried out for 5min at 72 ℃.

The results of the double cleavage assay showed that the product was a band of about 12kb in length and a band of about 1161bp in length (FIG. 2, 2). From this, it was found that the desired gene PfbZIP was ligated to the pCAMBIA1303 vector to obtain a pCAMBIA1303-PfbZIP52 recombinant plasmid, i.e., an overexpression vector of PfbZIP gene.

10. Sequence analysis

Sequencing recombinant plasmid bacterial liquid which is detected to be positive by electrophoresis to a Optimago technology (Western-style) Co., ltd, and comparing the sequencing result with a PfbZIP gene CDS sequence in a transcriptome, wherein the result shows that the CDS full length 1161bp of PfbZIP52 codes 386 amino acids, the molecular weight of the protein coded by the gene is 42.9kDa, the theoretical isoelectric point pI is 7.76, the NCBI-CDD functional domain prediction result shows that the protein contains 1 bZIP functional domain (bZIP_plant_BZIP 46) and is positioned between 292-346 amino acid residues, and the protein coded by the gene PfbZIP belongs to a member in bZIP super family superfamily (A in FIG. 3); pfbZIP52 gene CDS sequence contains 4 exons and 3 introns (B in fig. 3); pfbZIP52 the encoded protein contains 3 major conserved Motif motifs, motif 1, motif2 and Motif3 (C in FIG. 3).

SEQ ID NO.3：

ATGGATGGAGGTTGTGAGCAAGAGCAAGAGCAAATAGAGCTAATGAAGAACCAGAGTTGGACATCATCATCAATACTAGGGCGTCAATCGACGATATACTCATTGACTCTTGATGAATTCCAACACACACTTTGTGAGAGTGGGAAGAATTTTGGGTCAATGAACATGGACGAATTCCTCAATAGCATATGGACTGCTGAAGAAAATGAAGCTCAAGCAAGTAATGTCAATAATGGCATTAATGTGATGCAGCAGCAGTTCCCTCTGCAGGATAGTAGTAACAGTAATGGAAAGGGATTAATAGCTAAGCAACCTAGCTTGCCGAGACAGGGTTCTCTCAGCATCCCAGAGCCTCTTTGCAGGAAAACTGTGGACCAAGTGTGGTCTGAGATTCACAAGGATGAGCAGCACCACCACCTTCATACCACATCATCAGCTCAGAAACAGGCTACATTCGGAGAGATGACACTCGAAGATTTCTTGGTACGTGCCGGGGTTGTAAGAGAACAGAATTACCCACCTCAACCTCCACCTCCACCCCCACCTAATAATTTTGGAATGTTCCACAACAACAACAATAACCATCCTAATAGTAATTTTGTTGCAAGCCCTCCTGTTGCTGTTGGGATCAACATTAATGTTGGGGGTTATCAACATCATCACCAGCCCCTCGGTGTAGGAATGAAGAGGGGCAATGGGTACTCATCACAATCACAATCACAACCCCTTCCTCCGGCTGCAGCCAATGGAGGAGGAGGAGGTACAGGTGGTGGTTTTGGGATGGGGTCGCCAGTAAGTCCTTTGTCAGATGGAATGGGTGCCAGAGGAGGACGGAAGCGGATGTCGGATGGCCCCGTGGAGAAGGTGGTGGAGAGGCGACAACGAAGAATGATCAAGAACAGAGAGTCTGCCGCACGATCAAGGGCAAGGAAGCAGGCTTACACGGTGGAGCTCGAAGCAGAACTGAACCAACTCAAGGAAGAGAATTTACACCTGAAGCAAGCTCTGGCTGAATCAGAGAGGAAAAGAAAACAGCAGTACTACGAGGAAGAGGAGGCGAGACAGGCACATGAGGTGCAGCAGATGCAAACAAAGTCGTCGTCATACAAAGCTAATGACAAATTACGAGCAATGAGGAGGAGTTTCAGCTGTCCTTACTGA

Example 2: and (3) analyzing the expression of PfbZIP genes in different tissues and different stages of seed development of the perilla.

1. Experimental method

Taking perilla roots, stems, leaves, flowers and seeds (seed a, seed b, seed c and seed d) of 10, 20, 30 and 40d after flowering, extracting total RNA, performing reverse transcription to form a cDNA first chain, and performing real-time fluorescence quantitative PCR analysis by taking PfActin as an internal reference gene;

the primers used for amplification are: GCTCAAGCAAGTAATGTCAA (SEQ ID NO. 4), CTCCGAATGTAGCCTGTT (SEQ ID NO. 5).

As shown by the analysis of FIG. 4, pfbZIP gene is expressed in different tissues of Perilla frutescens, but the expression level in the seeds is highest, and the expression level gradually increases as the seeds of Perilla frutescens mature, which indicates that PfbZIP gene plays an important role in the synthesis and accumulation of oil in the seeds of Perilla frutescens.

Example 3: construction of transgenic plants.

1. Experimental materials

The heterologous expression host plant, agrobacterium tumefaciens strain GV3101, nicotiana tabacum (Nicotiana tabacum) variety Sumsun NN (SNN), was used, all supplied by the Shanxi university molecular agriculture and bioenergy research institute. The reagents used were 2 XE-TaqPCRMasterMix (Hangzhou New Biochemical development Co., ltd.), LB broth, LB nutrient agar, kanamycin (Kan), rifampicin (Rif), MS medium (without agar and sucrose), cephalosporin (Cef), hygromycin (Hyg), 6-benzylaminopterin (6-BA), alpha-naphthylacetic acid (NAA) (Beijing Soy Bao technology Co., ltd.).

2. Preparation of Agrobacterium GV3101 competent cells

Selecting a strain of Agrobacterium tumefaciens, plating on LB solid medium containing 50mg/mL rifampicin (Rif), and culturing at 28 ℃ in dark for about 48 hours; picking single colony, inoculating in 50mL LB liquid medium containing 50mg/mL Rif, culturing at 28 ℃ and 220rpm under shaking until OD ₆₀₀ is about 0.6; centrifugation at 5000rpm for 5min, discarding supernatant and resuspension of the cells with 10mL of 0.15mol/LNaCl solution (pre-chilled at 4 ℃); 5000rpm, centrifuging for 5min, discarding supernatant, and re-suspending the precipitate with 1/50 original volume of 20mmol/L CaCl ₂ (containing 15% glycerol) to prepare GV3101 competent.

3. Transformation of Agrobacterium GV3101 into pCAMBIA1303-PfbZIP52

3. Mu.g of pCAMBIA1303-PfbZIP recombinant plasmid was taken and added to 200. Mu.L of GV3101 competent cells; ice bath for 5min, and freezing in liquid nitrogen for 5min (or standing at-70deg.C for 10 min), and heat shock at 37deg.C for 5min; adding 800 mu L of LB liquid medium without antibiotics, and carrying out shake culture at a low speed (175 rpm) at 28 ℃ for 4 hours; 3000rpm, centrifuging for 4min, discarding supernatant, leaving about 200 μl of culture medium to resuspend bacterial pellet, sucking appropriate amount of bacterial liquid, and coating on LB plate medium containing corresponding antibiotics, and culturing at 28deg.C for about 48 hr.

4. PCR identification of recombinant plasmid pCAMBIA1303-PfbZIP for transforming Agrobacterium GV3101

Single colonies were picked and inoculated into 1.5mL centrifuge tubes with LB liquid medium (containing 50. Mu.g/mL Rif and 50. Mu.g/mL Kan), cultured at 28℃with shaking at 200rpm overnight. The PCR reaction was carried out using the cultured bacterial liquids as templates and SEQ ID NO.1 and SEQ ID NO.2 as detection primers in example 1, respectively, using 2 XE-TaqPCRMasterMix, and the PCR amplification system was 10. Mu.L as follows: PCR template 0.5. Mu.L, SEQ ID NO.1 and SEQ ID NO.2 primers each 0.2. Mu.L, 2 XE-TAQPCRMASTER MIX. Mu. L, ddH ₂ O4.1. Mu.L; the PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 80s,30 cycles; after 5min of extension at 72 ℃, the product is used for agarose gel electrophoresis detection, and the length of the product is 1161bp, which indicates that the transformation is successful.

The medium used for Agrobacterium GV3101 transformation was as follows:

LB liquid Medium 1L: LB broth 20g, dissolved in 1L distilled water. High temperature and high pressure steam sterilization at 121deg.C for 15min.

LB solid Medium 1L: LB nutrient agar 32g was dissolved in 1L of distilled water. High temperature and high pressure steam sterilization at 121deg.C for 15min.

5. Genetic transformation of tobacco

Aseptic tissue culture seedling of common tobacco: placing common tobacco seeds into a 1.5mL centrifuge tube, reversing and uniformly mixing with 70% alcohol for 40s, and flushing with sterile water for 3 times; mixing with 0.01% HgCl ₂ upside down for 6min, and washing with sterile water for 3 times; inoculating the sterilized tobacco seeds on a germination culture medium for culture, and placing the tobacco seeds in an illumination incubator for germination and growth; obtaining the aseptic tissue culture seedling of the tobacco after the tobacco seeds germinate and the true leaves grow out and grow stably;

Germination medium: 2.37g MS medium powder+30 g/L sucrose+7 g/L agar.

Tobacco leaf preculture: cutting leaves of sterile tissue culture seedling of common tobacco with bright green color and strong growth into blocks with the size of about 0.5cm ², and spreading on tobacco genetic transformation preculture medium for 2d.

Co-culturing tobacco leaves: the agrobacteria GV3101 bacteria liquid transformed with pCAMBIA1303-PfbZIP recombinant plasmid is inoculated in LB liquid culture medium (containing 50 mug/mLRif and 50 mug/mLKan) for culturing until OD ₆₀₀ reaches 0.8, and then the tobacco leaves are infected, thus obtaining the transgenic tobacco leaves. The specific process is as follows: immersing the tobacco leaves after preculture in the cultured agrobacterium GV3101 bacterial liquid, carrying out shaking infection for 8min, washing with sterile water for 1min, and spreading the tobacco leaves on a co-culture medium for dark culture for 2d to obtain the transgenic tobacco leaves.

Callus induction and screening culture: transferring the co-cultured transgenic tobacco leaves to a screening culture medium to induce callus differentiation until the cluster buds grow out.

Rooting culture: transferring the cluster buds into a rooting culture medium for further culture, identifying positive transgenic plants after the transgenic tobacco leaf seedlings grow out of root systems and grow stably, and performing seedling hardening and transplanting.

The media used for genetic transformation of tobacco were as follows:

Pre-culture medium: 4.74g MS medium powder+30 g/L sucrose+7 g/L agar+1.0 mg/L6-benzylaminopurine (6-BA) +0.1mg/L alpha-naphthylacetic acid (NAA).

Co-culture medium: 4.74g MS medium powder+30 g/L sucrose+7 g/L agar+1.0 mg/L6-benzylaminopurine (6-BA) +0.1mg/L alpha-naphthylacetic acid (NAA).

Screening the culture medium: 4.74g MS medium powder+30 g/L sucrose+7 g/L agar+1.0 mg/L6-benzylaminopurine (6-BA) +0.1mg/L alpha-naphthylacetic acid (NAA) +500mg/L cephalosporin (Cef) +5mg/L hygromycin (Hyg).

Rooting medium: 2.37g MS medium powder+30 g/L sucrose+7 g/L agar+500 mg/L cephalosporin (Cef).

6. Positive transgenic tobacco plant identification

Positive transgenic tobacco genome level identification: after the aseptic seedlings of the transgenic tobacco root and grow robustly, extracting DNA of the transgenic tobacco and Wild Tobacco (WT) as templates, taking SEQ ID NO.1 and SEQ ID NO.2 as detection primers, and carrying out PCR reaction by using 2 XE-TaqPCR MasterMix, wherein the PCR amplification system is 10 mu L as follows: PCR template 0.5. Mu.L, SEQ ID NO.1 and SEQ ID NO.2 primers each 0.2. Mu.L, 2 XE-Taq PCR MasterMix. Mu. L, ddH ₂ O4.1. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 80s,30 cycles; after 5min of extension at 72 ℃, the gel was used for agarose gel electrophoresis detection.

The agarose gel electrophoresis result shows that the product length is 1161bp, which indicates that PfbZIP gene is successfully transferred into tobacco (A of FIG. 5).

The PfbZIP gene was successfully introduced into tobacco, then translated and transcribed, the RNA of PfbZIP transgenic tobacco with positive genome level was extracted, and after reverse transcription into cDNA, PCR detection was performed from the transcription level, and the primer set in any region including the full-length coding region of PfbZIP gene was amplified to a partial region (250 bp) of PfbZIP gene.

Identification of positive transgenic tobacco transcript levels: RNA of transgenic tobacco and Wild Tobacco (WT) whose genome level detected positive was extracted, and cDNA was synthesized by reverse transcription, and PCR was performed using 2 XE-TaqPCRMasterMix with SEQ ID NO.4 and SEQ ID NO.5 as detection primers in example 2, and 10. Mu.L of PCR amplification system was as follows: PCR template 0.5. Mu.L, SEQ ID NO.4 and SEQ ID NO.5 primers each 0.2. Mu.L, 2 XE-TaqPCRMasterMix. Mu. L, ddH ₂ O4.1. Mu.L. The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 30s,30 cycles; after 5min of extension at 72 ℃, the gel was used for agarose gel electrophoresis detection.

The agarose gel electrophoresis result shows that the product length is 250bp, which indicates that PfbZIP gene can be effectively transcribed and expressed in tobacco plants (B of FIG. 5).

According to the method, agrobacterium tumefaciens GV3101 is used as a strain, and agrobacterium tumefaciens GV3101 competent cells are prepared; then transforming the competent cells of the agrobacterium GV3101 by using a recombinant plasmid pCAMBIA1303-PfbZIP to obtain the agrobacterium GV3101 containing pCAMBIA 1303-PfbZIP; tobacco is used as a heterologous host, and agrobacterium GV3101 containing pCAMBIA1303-PfbZIP is infected with tobacco to obtain a PfbZIP gene over-expression positive transgene type (OE) tobacco plant.

Example 4: functional verification of PfbZIP gene.

1. Experimental materials

PfbZIP52 gene overexpression positive transgenic (OE) tobacco plants and wild-type (WT) tobacco plants; the kit for quantitatively determining the vegetable protein (Nanjing established biological engineering institute), the kit for detecting the starch content of the vegetable (Beijing Soy Bao technology Co., ltd.) and the kit for detecting the soluble sugar content of the vegetable (Beijing Soy Bao technology Co., ltd.).

2. Determination and analysis of total lipid and main fatty acid component content of transgenic tobacco

And taking transgenic tobacco leaves, fully grinding the transgenic tobacco leaves into powder after vacuum freeze drying, extracting the transgenic tobacco leaves by a chloroform-methanol method, and measuring the total grease content of the transgenic tobacco leaves. 50mg of each sample was weighed and placed in a 50mL centrifuge tube, and 3 replicates were set for each sample. 7.5mL of chloroform-methanol (V: V=1:2) solution was added to each centrifuge tube, and after blowing and sucking, the mixture was placed in a shaking table at 37℃for shaking extraction for 24 hours, centrifuged at 4000rpm for 10 minutes, and the supernatant was carefully sucked into another new centrifuge tube. Chloroform was added to the remaining precipitate in 7.5mL of methanol solution and extraction was continued at 37℃for 12 hours. The two supernatants were combined, 9mL of 1% NaCl solution and 5mL of chloroform solution were added to give a final volume ratio of chloroform to methanol to water of 2:2:1.8, and after mixing well, the mixture was centrifuged at 4000rpm for 10min, and the lower organic phase was sucked into a clean finger-shaped tube (weighing m0 before use), and dried with a nitrogen purge and then weighed again (m 1). Total oil content = (m 1m 0)/0.05x100% for each sample.

The fatty acid methyl esters of tobacco leaves were prepared by the chloroform-methanol method, and the main fatty acid components and their contents in tobacco were measured using GC gas chromatograph (agilent, 7890B). The specific operation is as follows: separately, 50mg of the lyophilized powder of transgenic tobacco leaves was weighed into a clean finger tube, 50. Mu.L of Tri 17:0 alkanoic acid (10 mg/mL) was added and used as an internal standard control, and 3 replicates were used for each sample. Chloroform-methanol (V: v=2:1) solution 1mL was added and mixed by vortexing. Then, 500. Mu.L of 0.9% KCl solution and 1mL of chloroform solution were sequentially added thereto, and the mixture was vortexed and centrifuged at 4000rpm for 10 minutes. Sucking the lower organic phase into a new finger-shaped tube, adding 500 mu L of methanol (containing 2.5% of concentrated sulfuric acid) after the organic phase is completely evaporated, shaking and mixing uniformly, and then placing in a water bath kettle at 80 ℃ for reaction for 2 hours. After cooling, 1mL of 0.9% KCl solution and 500. Mu.L of n-hexane solution were added, and after mixing, the mixture was centrifuged at 4000rpm for 10min. Carefully aspirate the supernatant phase into a new finger-shaped tube, blow-dry with a nitrogen purge, add 500 μl of n-hexane again, dissolve the fatty acid precipitate thoroughly, aspirate into GC vials, store at-20 ℃ for subsequent fatty acid component and assay.

Determining main fatty acid components and content of perilla seeds by using a GC gas chromatograph, wherein the GC reaction procedure is as follows: the initial temperature is 50 ℃ for 0.5min, the temperature is increased to 194 ℃ according to 30 ℃/min, the operation is 3.5min, the temperature is increased to 240 ℃ according to 5 ℃/min, and the temperature is kept for 1min; the temperature of the FID detector is set to 280 ℃, the hydrogen gas is 40mL/min, the air is 400mL/min, and the tail gas is blown with nitrogen gas to 25mL/min; the sample injection amount was 1. Mu.L. The fatty acid components were identified by an internal standard method and the content thereof was calculated, and the content ki= (ai×ms)/(as×m) ×100% of each fatty acid component. Where i is the measured peak area of each fatty acid component, ai is the mass of the internal standard (Tri 17:0 alkanoic acid), as is the peak area of the internal standard (Tri 17:0 alkanoic acid), and m is the mass of the weighed sample.

As shown by the result A of FIG. 6, the total fat content of PfbZIP transgenic type (OE) tobacco is significantly higher than that of Wild Type (WT) tobacco, and as shown by the result B of FIG. 6, the over-expression of PfbZIP gene significantly increases the C18:3 and C16:0 contents of transgenic tobacco by 4.48% and 3.52%, respectively, while the C18:1 and C18:2 contents are reduced by 2.71% and 3.20%, respectively, without significant change in the C18:0 and C20:0 contents (C16:0 palmitic acid, C18:0 stearic acid, C18:1 oleic acid, C18:2 linoleic acid, C18:3 linolenic acid, C20:0 arachidic acid). The result shows that the PfbZIP gene is over-expressed in the tobacco, so that the total fat content of the transgenic tobacco is improved, the enrichment of fatty acid components of the transgenic tobacco and the content thereof are changed, and the PfbZIP gene is proved to play a role in the metabolism of vegetable fat.

3. Analysis of other agronomic traits in transgenic tobacco

Determination of soluble sugar content of transgenic tobacco: the soluble sugar content of the purple perilla transgenic tobacco is measured by using a plant soluble sugar content detection kit, and the specific operation is as follows: weighing 100mg of transgenic tobacco leaf samples which are freeze-dried and ground into powder, adding 1mL of deionized water, fully grinding to be in a uniform slurry state, transferring into a centrifuge tube with a cover, and reacting for 10min in a boiling water bath. After cooling to room temperature, centrifugation is carried out at 8000rpm for 10min, the supernatant is sucked into a10 mL test tube, and deionized water is fixed to a volume of 10mL. Sucking 40 mu L of sample liquid into a new centrifuge tube, adding 20 mu L of working liquid (in the conventional preparation, adding 5mL of reagent II into a bottle of reagent I to fully dissolve), uniformly mixing, and reacting for 10min in a water bath at 95 ℃. After cooling to room temperature, the absorbance A at 620nm was measured using a spectrophotometer with deionized water as a blank reference. The regression equation obtained from the standard curve was y=0.1329x+0.0085, where x represents the absorbance value and y represents the standard concentration (mg/mL). The calculation formula of the soluble sugar content of each sample is as follows: soluble sugar content (mg/g) = (y×v1)/(w×v1/V2). Wherein, V1: sample fluid volume, 40 μl; v2: the volume of the extracting solution is 10mL; w: sample mass, 0.1g.

Determination of transgenic tobacco total protein content: the protein content of the purple perilla developmental seeds is measured by using a plant protein quantitative measurement kit (built by Nanjing), and the specific operation is carried out according to the specification of the kit: weighing 100mg of transgenic tobacco leaf samples to be detected in a 1.5mL centrifuge tube, adding 9 times of 0.9% NaCl solution in volume into the centrifuge tube according to the proportion of 1:9 (mass: volume=g: mL), fully grinding the sample on ice to be in a uniform slurry state, uniformly mixing by vortex, centrifuging at 4000rpm for 10min, and carefully sucking the supernatant; the supernatant and 0.9% NaCl solution are diluted to 2% according to the ratio of 1:4 or diluted to 1% according to the ratio of 1:9, and the mixture is preserved for testing. Adding 0.05mL deionized water, 0.05mL protein standard solution (mL, 0.563 g/L) and 0.05mL sample homogenate into a blank tube, a standard tube and a measuring tube respectively, and adding 3mL coomassie brilliant blue color development solution into each tube; fully and uniformly mixing the reagents in each reaction tube, and standing for 10min at room temperature; the OD value in each reaction tube was determined by setting the spectrophotometer wavelength 595nm and using deionized water as a blank reference. Total protein content calculation of transgenic tobacco: sample protein concentration (g/L) = [ (OD measurement OD blank)/(OD standard OD blank) ] C standard.

Determination of starch content of transgenic tobacco: the starch content of the transgenic tobacco is measured by using a plant starch content detection kit (Soxhobao), and the specific operation is carried out according to the instruction of the kit: weighing 100mg of the transgenic tobacco leaf sample which is frozen and dried and ground into powder, putting the transgenic tobacco leaf sample into a 2mL centrifuge tube, adding 1mL of reagent, fully and uniformly mixing, putting the transgenic tobacco leaf sample into a water bath kettle, reacting for 0.5h at 80 ℃, centrifuging for 5min at 3000g, and discarding the supernatant; adding 500 mu L of sterile water into the residual sediment, and placing the mixture in a boiling water bath for gelatinization reaction for 15min; after cooling to room temperature, adding 350 mu L of a second reagent, and placing in a boiling water bath again for gelatinization reaction for 15min (during which the mixture is mixed for 3-5 times in a reverse way); cooling, adding 850 μl of sterile water, mixing, centrifuging at 8000g at room temperature for 15min, and collecting supernatant (sample solution) for subsequent detection (100 μl supernatant+700 μl sterile water); 200 mu L of sample liquid is sucked into a new centrifuge tube, 1mL of working liquid (in the existing preparation, 6.75mL of distilled water is firstly added into the reagent III, 38.25mL of concentrated sulfuric acid is then added to make the reagent III fully dissolved) is added into the centrifuge tube, and the mixture is uniformly mixed and placed into a water bath kettle to react for 10min at 95 ℃; after cooling to room temperature, the absorbance A at 620nm was measured using a spectrophotometer, with sterile water as a blank reference. The regression equation obtained from the standard curve is y=0.1031x+0.0009, where x represents the standard concentration (mg/mL) and y represents the absorbance value. Starch content calculation formula of each sample: starch content (mg/g) =x x dilution x V extraction/W. Wherein, V extracts: volume after extraction, 1.7mL; w: sample mass, 0.1g; dilution factor: 8 times.

As a result, as shown in fig. 7, transgenic (OE) tobacco plants had significantly increased soluble sugar content (a) compared to wild-type (WT) tobacco plants, no significant change in total protein content (B) of fig. 7, significantly decreased starch content (C) of fig. 7, and changes in tobacco soluble sugar and starch content indicated that overexpression of PfbZIP gene altered carbon flux into the lipid synthesis pathway in tobacco plants. In addition, the PfbZIP transgenic tobacco lines were analyzed for seed germination, seed thousand kernel weight, and leaf photosynthetic rate (Photosynthetic rate, pn), 3 replicates per sample. The results show that there is no significant difference in seed germination (D of fig. 7), seed thousand kernel weight (E of fig. 7) and leaf photosynthesis rate (F of fig. 7) of transgenic (OE) tobacco plants compared to Wild Type (WT) tobacco plants. Therefore, the result shows that the overexpression of PfbZIP gene promotes carbon flux in tobacco to enter the grease/FA synthesis path, and has the function of promoting the synthesis of tobacco grease.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (10)

1. The overexpression vector of the PfbZIP52 gene is characterized in that the nucleotide sequence of the PfbZIP gene is shown as SEQ ID NO.3, the overexpression vector is obtained by connecting a PfbZIP gene with a plant expression vector, the plant expression vector comprises Xba I and Kpn I enzyme cutting sites, the PfbZIP gene is connected between the Xba I and Kpn I enzyme cutting sites, and the overexpression vector can be used for PfbZIP gene function research and can improve the content of tobacco oil and fat by regulating fatty acid metabolism.
2. 2. The PfbZIP gene overexpression vector according to claim 1, wherein the plant expression vector is any one of pCAMBIA1303 vector, pCAMBIA1391Z vector, pCAMBIA1302 vector.
3. 3. The method for constructing the PfbZIP gene overexpression vector as defined in claim 2, comprising the steps of:

   Amplifying a target gene PfbZIP shown as SEQ ID NO.3 by PCR;

   Recovering and purifying the target gene PfbZIP to obtain a target gene PfbZIP52 fragment;

   The pCAMBIA1303 vector plasmid is subjected to double-enzyme tangential linearization, purification and recovery to obtain a pCAMBIA1303 vector linear fragment;

   connecting the recovered target gene PfbZIP fragment with the pCAMBIA1303 vector linearization fragment to obtain a recombinant product pCAMBIA 1303-PfbZIP;

   the recombinant product pCAMBIA1303-PfbZIP is transformed by DH5 alpha competent cells to obtain the overexpression vector of PfbZIP gene.
4. 4. The method according to claim 3, wherein the PCR amplification reaction system comprises: 2×FastPfu FlyPCR SuperMix. Mu.L, 1. Mu.L of cDNA template, 1. Mu.L each of primer pair PfbZIP-1303, no nuclease water make up to 50. Mu.L.
5. 5. A method of construction according to claim 3, wherein the system for ligating the recombinant product pCAMBIA1303-PfbZIP is: the target gene PfbZIP fragment 2 mu L, pCAMBIA1303 vector linearized fragment 3 mu L, 2X ClonExpress Mix mu L.
6. 6. Use of the PfbZIP gene over-expression vector of claim 1 in improving tobacco oil quality.
7. 7. The use according to claim 6, wherein the nucleotide sequence of PfbZIP gene is shown as SEQ ID NO.3, the over-expression vector is obtained by connecting PfbZIP gene with plant expression vector, the plant expression vector comprises Xba I and Kpn I enzyme cutting sites, the PfbZIP gene is connected between the Xba I and Kpn I enzyme cutting sites, the over-expression vector is transferred into tobacco construction to obtain transgenic tobacco, and the content of tobacco fat is improved by regulating fatty acid metabolism.
8. 8. The use according to claim 7, characterized in that the construction step of the transgenic tobacco is as follows:

   Constructing PfbZIP gene over-expression vector;

   Transforming the PfbZIP gene over-expression vector into agrobacterium GV3101 to obtain agrobacterium GV3101 bacterial liquid;

   culturing tobacco leaves on a tobacco genetic transformation preculture medium;

   Infecting the cultured tobacco leaves with agrobacterium GV3101 bacterial liquid to obtain transgenic tobacco leaves;

   transferring the transgenic tobacco leaves to a screening culture medium for culture until cluster buds grow out;

   transferring the cluster buds into a rooting culture medium for culture, and obtaining positive transgenic tobacco plants after the transgenic tobacco leaves grow out of root systems.
9. 9. The use according to claim 8, wherein the pre-culture medium formulation is 4.74gMS ms medium powder+30 g/L sucrose+7 g/L agar+1.0 mg/L6-ba+0.1mg/LNAA;

   the co-culture medium formula is 4.74g MS culture medium powder, 30g/L sucrose, 7g/L agar, 1.0 mg/L6-BA and 0.1mg/LNAA.
10. 10. The use according to claim 8, wherein the screening medium formulation is 4.74g MS medium powder+30 g/L sucrose+7 g/L agar+1.0 mg/L6-ba+0.1 mg/lnaa+500mg/LCef +5mg/L Hyg;

    the rooting culture medium is 2.37g of MS culture medium powder, 30g/L of sucrose, 7g/L of agar and 500mg/LCef.