CN112626096A - Method for creating transgenic plant with high palmitic acid content and multiple leaves and application - Google Patents

Abstract

The invention relates to a method for creating high palmitic acid and multi-leaf transgenic plants by using fatty acyl-acyl carrier protein thioesterase AhFATB2 gene and application thereof, wherein the method comprises the following steps: (1) cloning of the arachidic acyl-acyl carrier protein thioesterase Gene: extracting peanut seed RNA by using Trizol, and amplifying AhFATB2 gene by using cDNA reverse transcribed by the RNA as a template; (2) construction of AhFATB2 Gene plant overexpression vector pBI-AhFATB 2: replacing GUS gene fragment on the plasmid pBI121 by the cloned AhFATB2 gene fragment; (3) genetic transformation of pBI-AhFATB2 in Arabidopsis thaliana to obtain transgenic positive plants. The invention takes wild plants as transgenic receptors, and artificially creates transgenic plants by over-expressing AhFATB2 genes. The method can improve the content of palmitic acid in plant seeds and the biomass of leaves of the plant seeds, and has certain development value.

Description

Method for creating transgenic plant with high palmitic acid content and multiple leaves and application

Technical Field

The invention relates to a method for creating high palmitic acid and multi-leaf transgenic plants by using fatty acyl-acyl carrier protein thioesterase AhFATB2 gene and application thereof, belonging to the technical field of plant genetic engineering.

Background

During fatty acid synthesis, fatty acyl-acyl carrier protein thioesterases (Fat) determine fatty acid chain length by catalyzing the hydrolysis of thioester bonds. Fats are divided into two classes, FatA and FatB, according to the sequence and substrate specificity, and constitute the TE14 thioester-active enzyme gene family. FatA plays a specific role in the synthesis of unsaturated long-chain fatty acyl-acyl carrier proteins, especially during oleoyl-coa (oleoyl-ACP) synthesis, but is less active during saturated fatty acyl-acyl carrier protein synthesis. FatB is a saturated acyl-acyl carrier protein mainly generated by taking 8-18 carbon chains as substrates. FatBs can be further divided into FatB1 and FatB2 subclasses, FatB1 exists in all plants, mainly with long-chain saturated acyl-ACP as substrate, especially 16: 0-ACP; FatB2 is only present in plants that accumulate 8-14 carbon chain fatty acids, primarily with short and medium chain saturated fatty acids as substrates. Fat substrate specificity is crucial for fatty acid component regulation, so the Fat component is determined by the Fat coding gene.

Fat genes with 8-18 carbon chains as substrates in various plants have been cloned at present, and a plurality of long-chain specific FatB genes are verified to have functions through transgenes. Overexpression of GarmFatA1, MlFatB and AtFATA2 resulted in an increase in stearic acid (18:0) content, overexpression of AtFatB1, BnFatB (2), overexpression of CpFatB2a in tobacco resulted in a 46% increase in myristic acid content (14:0) in leaves, while overexpression of ClFatB1 and GmFATA1 increased palmitic acid (16:0) and stearic acid (18:0) content simultaneously. JtFatB1, AtFatB increased the palmitic acid (16:0) content mainly. Overexpression of UcFatB1 in canola resulted in a negligible increase in lauric acid (12:0) content to nearly 60%, overexpression of CpFatB2a in tobacco resulted in a 46% increase in myristic acid content (14:0) in leaves, while overexpression of ClFatB1 and GmFATA1 increased both palmitic acid (16:0) and stearic acid (18:0) content.

Palmitic acid (Palmitic acid), a saturated higher fatty acid, is commonly present in animal and vegetable fats and oils in the form of glycerides, mainly derived from palm oil and palm kernel oil. Palmitic acid does not contain C ═ C double bonds, belongs to saturated fatty acid non-essential fatty acid, is related to growth and development of children and growth health, has the treatment effects of reducing blood fat, preventing and treating coronary heart disease and the like, and has a certain relation with physiological functions of intelligence development, memory and the like. The esterification reaction of palmitic acid can produce biodiesel, which proves that the palmitic acid can be used as raw material oil of the biodiesel, and the palmitic acid ester is a typical fatty acid ester, can be used as a raw material or an intermediate of a plasticizer, a surfactant, a fine chemical product and the like, and is widely used for manufacturing agriculture, food, medicines and fine chemical products. The southeast Asia countries utilize the superior climatic conditions to plant a large amount of palmitic acid to obtain the palmitic acid which is used as the raw material of the biodiesel. Therefore, the palmitic acid has wide application value in food processing and industrial production.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for creating high palmitic acid and multi-leaf transgenic plants by using a fatty acyl-acyl carrier protein thioesterase AhFATB2 gene and application thereof. The method of the invention uses wild plants as transgenic receptors, applies fatty acyl-acyl carrier protein thioesterase (AhFATB2) genes by means of genetic engineering, and artificially creates transgenic plants by over-expressing the AhFATB2 genes. The transgenic plant can improve the content of palmitic acid in plant seeds and the biomass of leaves of the plant seeds, and has certain development value.

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

a fatty acyl-acyl carrier protein thioesterase AhFATB2 gene, wherein the sequence of the AhFATB2 gene is shown in SEQ ID NO. 3.

A method for creating a transgenic plant by using a fatty acyl-acyl carrier protein thioesterase AhFATB2 gene is characterized in that a wild-type plant is used as a transgenic receptor, a gene engineering means is adopted, the fatty acyl-acyl carrier protein thioesterase AhFATB2 gene is used, and the transgenic plant is created artificially by over-expressing the AhFATB2 gene; the sequence of the AhFATB2 gene is shown in SEQ ID NO. 3.

The method for creating the transgenic plant comprises the following steps:

(1) cloning of the arachidic acyl-acyl carrier protein thioesterase AhFATB2 gene;

(2) constructing a plant over-expression vector pBI-AhFATB2 of the AhFATB2 gene;

(3) genetic transformation and positive seedling screening of pBI-AhFATB2 in Arabidopsis thaliana to obtain transgenic plant.

The cloning method of the AhFATB2 gene comprises the following steps: extracting peanut seed RNA by using Trizol, carrying out amplification of AhFATB2 gene by using cDNA reverse transcribed from RNA as a template, and using primers of AhFATB2S and AhFATB 2A;

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′。

the reaction system for AhFATB2 gene amplification is 25 mu L of reaction system containing: 1 XPCRbuffer, MgCI 1.5mmol/L, dNTP0.2mmol/L, primer concentration 0.5mol/L, Pfu enzyme 1.5 units, template 100 ng;

the PCR procedure was: 94 ℃ for 5 min; 94 ℃, 45s, 57 ℃, 45s, 72 ℃, 2min, 33 cycles; 72 ℃ for 10 min.

The construction method of the over-expression vector pBI-AhFATB2 comprises the following steps: replacing the GUS gene fragment on the plasmid pBI121 by the AhFATB2 gene fragment cloned in the step (1).

The specific method for constructing the over-expression vector pBI-AhFATB2 comprises the following steps: carrying out double enzyme digestion on a GUS fragment of the cloning vector pBI121 by using BamHI and SacI, and simultaneously carrying out enzyme digestion on the AhFATB2 gene fragment cloned in the step (1) by using BamHI and SacI; the restriction enzyme products of AhFATB2 gene fragments and the fragments cut from the cloning vector pBI121 are mixed, recovered by a DNA gel recovery kit, and then added with 5 units of T4 DNA ligase and 10 multiplied reaction buffer solution, the volume is supplemented to 20 mu L by sterile water, the mixture is connected overnight at 16 ℃, and the recombinant plasmid pBI-AhFATB2 is obtained after conversion.

The specific method of the step (3) is as follows: preparing agrobacterium tumefaciens GV3101 bacterial liquid containing recombinant plasmid pBI-AhFATB2, transforming plants, screening to obtain transgenic positive seedlings of the recombinant plasmid pBI-AhFATB2 successfully transferred into the plants, selfing the positive seedlings for more than 3 generations to obtain homozygous plants HF-AhFATB 2.

The screening method of the positive seedlings comprises the following steps: when the leaves grow to 3-4 leaf stage, taking green seedling leaves to carry out PCR positive detection, wherein the primers are as follows:

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′；

the PCR reaction system is as follows: genomic DNA template 1. mu.L, 10 XTaq enzyme reaction buffer 2uL, 25mM MgCL₂1.2uL, 2mM dNTP 1.5uL, 10uM primers of 0.2uL and 0.3 unit Taq enzyme respectively, and sterile water is added to 20 uL;

the reaction procedure is as follows: denaturation at 94 deg.C for 5 min; 94 ℃ for 45 s; 45s at 55 ℃; 72 ℃ for 2 min; 32 cycles, extension at 72 ℃ for 5 min.

The fatty acyl-acyl carrier protein thioesterase AhFATB2 gene is applied to improving the palmitic acid content of oil crop seeds and improving the biomass of plant leaves.

The invention has the beneficial effects that:

the invention provides a method for successfully cloning fatty acyl-acyl carrier protein thioesterase AhFATB2 gene, and application proves that the gene can be applied to transgenic plants, can improve the content of palmitic acid in plant seeds and the leaf biomass thereof, and has certain development value.

The invention takes wild plants as transgenic receptors, and artificially creates transgenic plants by over-expressing AhFATB2 genes. The AhFATB2 gene is overexpressed by a transgenic technology, the effect of the gene in plant development and seed palmitic acid synthesis is researched, and the gene has the functions of improving the plant palmitic acid content and increasing the plant leaf biomass. Meanwhile, the over-expression of the gene can not cause the change of other visible plant phenotypes and can not cause plant phenotypic defects.

The AhFATB2 gene in the peanut can be applied to plant genetic engineering, oil crop seed production and leaf generation of crops such as vegetables and tobacco, and is used for improving the content of palmitic acid in plant seeds and the leaf biomass thereof. The AhFATB2 gene expression can be increased or enhanced by artificially constructing an AhFATB2 overexpression vector or other genetic engineering and molecular biological means, and plants with high palmitic acid content and high leaf biomass are artificially created, so that the method has certain development value. If the gene is used for artificially creating plant materials with certain excellent agronomic traits and developing oil crops with high palmitic acid content, such as oil crops of rape, peanut, soybean and the like with high palmitic acid content, so as to meet various commercial requirements; the gene can also be used for artificially creating leafy plant materials, such as developing crops for harvesting leafy crops, such as tobacco, forage grass or vegetables, so as to meet different requirements.

Drawings

FIG. 1 is an electrophoretogram of a peanut AhFATB2 gene clone;

wherein: lane 1 shows the PCR amplification result using the DNA of peanut seeds as a template; lane 2 is the PCR amplification result using cDNA of peanut seeds as template; lane M is nucleic acid Marker;

FIG. 2 is a schematic diagram of a pBI-AhFATB2 vector constructed;

FIG. 3 is a PCR identification chart of a transgenic plant transformed with pBI-AhFATB 2;

wherein: m, DNAmarker DL 2000; lane 1, 2, 3, 4, 5, 6, 7, 8, positive strain; lane CK, wild type arabidopsis; p, plasmid control;

FIG. 4 shows the leafy phenotype of progeny of a plant overexpressing AhFATB2 gene;

wherein: a, 1 leaf of a transgenic plant; b, transgenic plant 2 leaves; c, wild type Arabidopsis thaliana leaves;

FIG. 5 is a descendant seed fatty acid component analysis of a plant over-expressing AhFATB2 gene;

wherein: a, analyzing oil components of the transgenic plant 1 seeds; b, analyzing the seed oil component of the transgenic plant 2; c, analyzing the oil component of wild arabidopsis thaliana seeds.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Example 1:

cloning of the arachidic acyl-acyl carrier protein thioesterase (AhFATB2) gene:

0.05-0.1g of cotyledon in the seed at any stage between 40 and 70 days after flowering of peanut was ground in liquid nitrogen to powdery and subjected to RNA extraction according to the requirements of Trizol extraction kit (purchased from Invitrogen). The extracted total RNA was dissolved in 60uL of double distilled water without RNase. DNase I was used to remove any residual DNA. The protein detector (DU 650BECKMAN, USA) is used for detecting the light absorption values of the RNA at 260 nm and 280 nm respectively, and the purity and the concentration of the RNA are identified by combining 1% (mass-to-volume ratio) agarose gel electrophoresis.

Reverse transcription was performed using the RNA obtained above as a template according to the following protocol: mu.L Oligo (dT) was added to 2. mu.g RNA, incubated at 70 ℃ for 5min, immediately placed on ice for 5min, briefly centrifuged, added with 4. mu.L 5 XM-MLVBuffer, 1. mu.L dNTP (10mmol/L), 20 units RNase Inhibitor, 200 units M-MLV reverse transcriptase (purchased from Promega), supplemented to a total volume of 20. mu.L with DEPC (diethyl pyrocarbonate) -treated sterile water, mixed well, incubated at 42 ℃ for 1h, water-washed at 70 ℃ for 15min, and the resulting cDNA was stored at-20 ℃ after being dispensed for further use.

The full-length cloning of AhFATB2 gene was performed using peanut seed cDNA as a template. Designing a primer according to the gene sequence of the peanut AhFATB2, wherein the primer comprises the following components:

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′(SEQ ID NO.1)；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′(SEQ ID NO.2)，

BamHI and SacI restriction enzymes were introduced into the 5' ends of the primer sequences, respectively.

The reaction system is 25 μ L of inclusion: 1 XPCR buffer, MgCI 1.5mmol/L, dNTP0.2mmol/L, primer concentration 0.5mol/L, Pfu enzyme 1.5 unit, template 100 ng.

The PCR procedure was: 94 ℃ for 5 min; 94 ℃, 45s, 57 ℃, 45s, 72 ℃, 2min, 33 cycles; 72 ℃ for 10 min.

The PCR reaction product was electrophoresed on 1% (mass/volume) low melting point agarose, and the results are shown in FIG. 1.

Cutting an amplification product strip from the gel, putting the amplification product strip into a 1.5ml Eppendorf centrifuge tube, carrying out water bath at 65 ℃ for 15min, adding equal volume phenol (PH7.9), shaking up for 5min in an inverted manner, centrifuging for 8 min at 13000 r/min, taking the supernatant, adding a mixture of equal volume chloroform and isoamyl alcohol (the volume ratio of chloroform to isoamyl alcohol is 24:1), shaking up for 5min in an inverted manner, centrifuging for 8 min at 13000 r/min, taking the supernatant, adding 1/10 volume of 3mol/L sodium acetate (PH5.2) solution and 2 volume of precooled 95% ethanol, mixing uniformly, putting the mixture into a refrigerator at-20 ℃ for more than 20min, centrifuging for 15min at 13000 r/min, pouring off 95% ethanol, then washing and precipitating with 75% ethanol, naturally air-drying, and dissolving DNA precipitate in 20 mul sterile deionized water. The desired fragment was recovered and purified to obtain AhFATB2 gene. The gene sequence is shown in SEQ ID NO. 3.

Example 2:

construction of plant overexpression vector pBI-AhFATB2 of AhFATB2 Gene and transformation of Agrobacterium tumefaciens strain GV 3101:

the constructed recombinant vector pBI-AhFATB2 was obtained by replacing the GUS gene on the plasmid pBI121 with the AhFATB2 gene fragment obtained by early cloning, and the vector map was as shown in FIG. 2.

First, the GUS fragment of the cloning vector pBI121 was double-digested with BamHI and SacI, while the AhFATB2 gene fragment was digested with BamHI and SacI. The cleavage reaction was carried out in a 37 ℃ incubator, and after about 4 to 6 hours, it was detected by electrophoresis on 1% agarose gel.

The enzyme-cleaved product of AhFATB2 gene fragment and the large fragment excised from the cloning vector pBI121 were recovered by using a DNA gel recovery kit. The digested product of AhFATB2 gene fragment was mixed with pBI121 vector at a ratio of 3:1 (wherein, 150ng of the digested product of AhFATB2 gene fragment and 50ng of pBI121 vector fragment) and then T4 DNA ligase 5 units, 10 Xreaction buffer, sterile water was added to make up a volume of 20. mu.L, and ligation was performed overnight at 16 ℃.

After transformation, pBI-AhFATB2 was transferred to Agrobacterium tumefaciens GV3101 (purchased from Shanghai Biotech Co., Ltd.) by freeze-thaw method, double-resistant plate screening was performed on a solid LB plate containing kanamycin (50. mu.g/mL) and rifampicin (50. mu.g/mL), colony PCR was performed by spotting, BamHI and SacI were verified by double-incision digestion, and the correctly verified recombinant plasmid was named pBI-AhFATB 2.

Example 3:

genetic transformation and transgenic plant screening of pBI-AhFATB2 in Arabidopsis thaliana:

agrobacterium tumefaciens GV3101 strain containing the constructed vector pBI-AhFATB2 was prepared, transferred to LB liquid medium containing 50. mu.g/ml kanamycin and 50. mu.g/ml rifampicin one day before transformation, and cultured overnight at 28 ℃. The next day, the absorbance was measured at 276nm using an ultraviolet spectrophotometer (SPEKOL 1300), and the bacterial solution was removed when the absorbance reached 1.6-2.0. The mixture was centrifuged at 4000g for 10min at room temperature (20-25 ℃ C.), the supernatant was discarded and the pellet was suspended in an equal volume of 5% sucrose (by mass/volume). The cloudy sucrose solution was poured into a large petri dish and Silwet l-77 (purchased from the Yuan Korea center, Wuzhou, Beijing) was added to a final concentration of 0.02% (by volume) prior to transformation.

After mixing evenly, the whole inflorescence of the arabidopsis to be transformed is slightly immersed in the sucrose for 15s, and the plant is taken out. The transformed plants were wrapped in a black plastic bag and cultured in a growth chamber. The plastic bag is uncovered the next day and placed in a place with light intensity for culture. The transformation is performed once more every other week. The seeds are harvested after about one month of culture, and the seeds are dried in an incubator or in the sun for 3-5 days.

The T0 generation seeds obtained by transformation and harvest are disinfected for 10 minutes by 70 percent (volume ratio) of alcohol and 0.01 percent (volume ratio) of mercury bichloride, washed for several times (5 to 7 times) by distilled water, and then evenly blown to the surface of MS solid screening culture medium. (MS macroelement mother liquor 100 ml; MS microelement mother liquor 10 ml; MS organic mother liquor 10 ml; MS iron salt 10 ml; inositol 10 ml; sucrose 30 g; pH is adjusted to 5.8 with 1M NaOH, 12g agar powder, constant volume is 1L, high pressure 121 ℃ sterilization is carried out for standby application; 8g agar powder is added to prepare a solid culture medium; various mother liquor formulas are shown in Table 1).

TABLE 1 MS culture Medium mother liquor formula

Vernalizing at 4 deg.C for 4-6 days, and culturing in constant temperature incubator. Positive shoots were selected based on the unique kanamycin resistance on the expression vector. When the leaves grow to be large enough (3-4 leaf stage), taking a little green seedling leaves to carry out PCR positive detection, wherein the primers are as follows:

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′；

the PCR reaction system is as follows: genomic DNA template 1. mu.L (about 50ng), 10 XTaq enzyme reaction buffer 2ul, 25mM MgCL₂1.2ul, 2mM dNTP 1.5ul, 10uM primers each 0.2ul, 0.3 unit Taq enzyme, sterile water to 20. mu.l.

The reaction procedure is as follows: denaturation at 94 deg.C for 5min, 32 cycles at 94 deg.C for 45s, 55 deg.C for 45s, and 72 deg.C for 2min, and extension at 72 deg.C for 5 min.

The PCR reaction product was detected by 1% agarose gel electrophoresis, and the result showed that the AhFATB2 gene expression vector pBI-AhFATB2 had been successfully transferred into Arabidopsis thaliana to obtain transgenic positive seedlings (FIG. 3). Selfing the positive seedlings for more than 3 generations to obtain a homozygous plant HF-AhFATB 2.

Example 4:

HF-AhFATB2 rosette number and palmitic acid content determination results:

the obtained homozygous plant HF-AhFATB2 seeds (transformants 1 to 10) were sown in vermiculite, covered with plastic cloth, vernalized at 4 ℃ and germinated in a growth chamber. Thinning after 2-3 true leaves grow out, so that the row spacing and the plant spacing are both 3 cm, and pouring the nutrient solution once every two weeks for vermiculite. Wild type plants 1-3 were also sown for comparison.

About 20 days later, after the arabidopsis plants have inflorescences, the number of rosette leaves is counted, the number of HF-AhFATB2 rosette leaves is 19-21, the number of wild type arabidopsis rosette leaves is 12-13, the sizes of the leaves are not obviously changed, and the biomass is obviously increased (figure 4, table 2).

After the arabidopsis thaliana is planted and matured, the palmitic acid content in the seeds is increased from 7.8% of the wild type to 19.2-28.8% and increased by 146-269% (table 3). As can be seen from the fatty acid component analysis (fig. 5): the areas of the peaks (corresponding to the values of 12.882, 12.893 and 12.881, respectively) representing the palmitic acid content in the fatty acid gas chromatogram of the transgenic plant seeds were significantly larger, and the peak areas changed from 7.85% of the wild type (12.881) to 22.49% of the seeds of the transgenic plant 1 (12.882) and 28.65% of the seeds of the transgenic plant 2 (12.893). The results show that the leaf number and the palmitic acid content of the transgenic plants obtained by the method are obviously increased.

TABLE 2 statistical Table of the number of rosette leaves transformed by HF-AhFATB2

TABLE 3 statistics of palmitic acid content of HF-AhFATB2 transformants

Therefore, the AhFATB2 gene of the peanut can be applied to plant genetic engineering, oil crop seed production and leaf formation of crops such as vegetables and tobacco, and has certain application value. Through artificially constructing an AhFATB2 overexpression vector or other genetic engineering and molecular biological means, the expression of the AhFATB2 gene is increased or enhanced, and plants with high palmitic acid content and high leaf biomass are artificially created for commercial production. For example, AhFATB2 is over-expressed in oil crops such as rape, peanut, soybean and the like, so that oil crops such as rape, peanut, soybean and the like with high palmitic acid are created and are used for commercially generating palmitic acid; AhFATB2 is overexpressed in crops for leaf harvesting such as tobacco, cabbage, and spinach to create leafy materials.

Sequence listing

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Claims (10)

1. A fatty acyl-acyl carrier protein thioesterase AhFATB2 gene, wherein the sequence of the AhFATB2 gene is shown as SEQ ID NO. 3.

2. A method for creating a transgenic plant by using a fatty acyl-acyl carrier protein thioesterase AhFATB2 gene is characterized in that a wild plant is used as a transgenic receptor, a gene engineering means is adopted, the fatty acyl-acyl carrier protein thioesterase AhFATB2 gene is used, and the transgenic plant is created artificially by over-expressing the AhFATB2 gene; the sequence of the AhFATB2 gene is shown in SEQ ID NO. 3.

3. The method of creating a transgenic plant of claim 2, comprising the steps of:

(1) cloning of the arachidic acyl-acyl carrier protein thioesterase AhFATB2 gene;

(2) constructing a plant over-expression vector pBI-AhFATB2 of the AhFATB2 gene;

(3) genetic transformation and positive seedling screening of pBI-AhFATB2 in Arabidopsis thaliana to obtain transgenic plant.

4. The method for creating a transgenic plant according to claim 3, wherein the AhFATB2 gene is cloned by: extracting peanut seed RNA by using Trizol, carrying out amplification of AhFATB2 gene by using cDNA reverse transcribed from RNA as a template, and using primers of AhFATB2S and AhFATB 2A;

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′。

5. the method for creating a transgenic plant according to claim 4, wherein the AhFATB2 gene is amplified in a reaction system of 25 μ L containing: 1 times PCR buffer, MgCI 1.5mmol/L, dNTP0.2mmol/L, primer concentration 0.5mol/L, Pfu enzyme 1.5 unit, template 100 ng;

the PCR procedure was: 94 ℃ for 5 min; 94 ℃, 45s, 57 ℃, 45s, 72 ℃, 2min, 33 cycles; 72 ℃ for 10 min.

6. The method for creating a transgenic plant according to claim 3, wherein the overexpression vector pBI-AhFATB2 is constructed by the following method: replacing the GUS gene fragment on the plasmid pBI121 by the AhFATB2 gene fragment cloned in the step (1).

7. The method for creating a transgenic plant according to claim 6, wherein the specific method for constructing the overexpression vector pBI-AhFATB2 comprises the following steps: carrying out double enzyme digestion on a GUS fragment of the cloning vector pBI121 by using BamHI and SacI, and simultaneously carrying out enzyme digestion on the AhFATB2 gene fragment cloned in the step (1) by using BamHI and SacI; the restriction enzyme products of AhFATB2 gene fragments and the fragments cut from the cloning vector pBI121 are mixed, recovered by a DNA gel recovery kit, and then added with 5 units of T4 DNA ligase and 10 multiplied reaction buffer solution, the volume is supplemented to 20 mu L by sterile water, the mixture is connected overnight at 16 ℃, and the recombinant plasmid pBI-AhFATB2 is obtained after conversion.

8. The method for creating a transgenic plant according to claim 3, wherein the specific method of step (3) is: preparing agrobacterium tumefaciens GV3101 bacterial liquid containing recombinant plasmid pBI-AhFATB2, transforming plants, screening to obtain transgenic positive seedlings of the recombinant plasmid pBI-AhFATB2 successfully transferred into the plants, selfing the positive seedlings for more than 3 generations to obtain homozygous plants HF-AhFATB 2.

9. The method of creating a transgenic plant of claim 8, wherein the positive shoot is selected by: when the leaves grow to 3-4 leaf stage, taking green seedling leaves to carry out PCR positive detection, wherein the primers are as follows:

AhFATB2S:5′-CGCGGATCCATGGTTGCTACTGCTGCTACG-3′；

AhFATB2A:5′-GACGAGCTCTCAGTTTTCTGCTGGAAAAACC-3′；

the PCR reaction system is as follows: genomic DNA template 1. mu.L, 10 XTaq enzyme reaction buffer 2uL, 25mM MgCL₂1.2uL, 2mM dNTP 1.5uL, 10uM primers of 0.2uL and 0.3 unit Taq enzyme respectively, and sterile water is added to 20 uL;

the reaction procedure is as follows: denaturation at 94 deg.C for 5 min; 94 ℃ for 45 s; 45s at 55 ℃; 72 ℃ for 2 min; 32 cycles, extension at 72 ℃ for 5 min.

10. Use of the fatty acyl-acyl carrier protein thioesterase AhFATB2 gene of claim 1 for increasing oil crop seed palmitic acid content and increasing plant leaf biomass.

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