CN112812160A

CN112812160A - Protein related to increasing oil content of plant, coding gene and application thereof

Info

Publication number: CN112812160A
Application number: CN202010037247.6A
Authority: CN
Inventors: 崔宇鹏; 于霁雯; 贾冰; 裴文锋; 吴嫚; 马建江; 张金发
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2020-01-14
Filing date: 2020-01-14
Publication date: 2021-05-18

Abstract

The invention discloses a protein related to the improvement of the oil content of plants, and a coding gene and application thereof. The invention also discloses an application of the protein in any one of the following m1) -m 5): m1) regulating and controlling the content of triglyceride of yeast; m2) regulating and controlling the content of vegetable oil; m3) constructing a recombinant yeast with increased triglyceride production; m4) cultivating transgenic plants with improved oil content; m5) plant breeding. The invention provides a protein related to the oil content of plant seeds and a coding gene thereof, which have important theoretical and practical significance for further clarifying the molecular mechanism of the oil content of the plant seeds and cultivating new varieties of crops with high oil content of the seeds by using a genetic engineering technology and means.

Description

Protein related to increasing oil content of plant, coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, particularly relates to a protein related to plant oil content improvement, a coding gene and application thereof, and particularly relates to a protein derived from cotton and related to plant oil content improvement, a coding gene and application thereof.

Background

Cotton is an important fiber and oil crop in the world. The cotton planting area of China all year round is about 600 million hectares, the ginned cotton is about 760 million tons, and the cotton seed is 1300 million tons. The cotton seeds are used as main byproducts of cotton production, about 1.65kg of cotton seeds can be produced according to the production of 1kg of lint, and the yield is huge; the cottonseed kernel after shelling accounts for 55% -60% of the weight of the seed, wherein the oil content is 30% -40%. The cottonseed oil is used as edible oil, is rich in essential fatty acid of a human body, has the linoleic acid content of about 55 percent, and is beneficial to reducing the occurrence of cardiovascular diseases of the human body; the cottonseed oil can also be used for producing biodiesel, wherein 99 percent of the carbon chain length of fatty acid is concentrated in C16 and C18, the conversion rate is up to more than 95 percent, and the cottonseed oil is sulfur-free, oxygen-rich and completely combusted without polluting the environment.

The cotton planting in China is centralized, the yield is stable, besides the cotton fiber can be put into the textile production, the cottonseed oil can be used for relieving the shortage of edible vegetable oil in China, and can also provide raw materials for the production of biodiesel, thereby being beneficial to environmental protection.

Disclosure of Invention

The first purpose of the invention is to provide a new application of GhGPAT protein or biological material related to the GhGPAT protein.

The invention provides application of GhGPAT protein or biological materials related to the GhGPAT protein in any one of the following m1) -m 5):

m1) regulating and controlling the content of triglyceride of yeast;

m2) regulating and controlling the content of vegetable oil;

m3) constructing a recombinant yeast with increased triglyceride production;

m4) cultivating transgenic plants with improved oil content;

m5) plant breeding.

In the application, the GhGPAT protein is derived from Gossypium hirsutum L., and is a protein shown in a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 3;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 3;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 3;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 3 and having the same function.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 3 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein GhGPAT in c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein GhGPAT in the step c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein GhGPAT in c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 at the 5 'end and/or 3' end.

In the above application, the biological material related to the GhGPAT protein is any one of the following a1) to A8):

A1) a nucleic acid molecule encoding a GhGPAT protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).

Further, the nucleic acid molecule of A1) is a gene as shown in 1) or 2) or 3) as follows:

1) the coding sequence is a cDNA molecule shown in a sequence 1 or a genome DNA molecule shown in a sequence 2;

2) a cDNA molecule or a genome DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by 1) and codes GhGPAT protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes GhGPAT protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc. In the present invention, sequence 1 in the sequence table is composed of 1503 deoxynucleotides, is the full-length cDNA sequence of GhGPAT protein, and is an open reading frame from position 1 to position 1503. The sequence 2 in the sequence table is a GhGPAT genomic DNA molecule, has the full length of 2339bp, and consists of 2 exons (1-618 th site and 1455-2339 th site) and 1 intron (619-1454 th site).

The nucleotide sequence encoding GhGPAT of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of GhGPAT isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode GhGPAT and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 3 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The above stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding GhGPAT (GhGPAT gene expression cassette) described in a2) refers to a DNA capable of expressing GhGPAT in a host cell, and the DNA may include not only a promoter that initiates transcription of GhGPAT but also a terminator that terminates transcription of GhGPAT. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator.

The recombinant vector containing the GhGPAT gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. In one embodiment of the present invention, the recombinant vector may be a recombinant expression vector pYES2-GhGPAT obtained by inserting the GhGPAT gene into the multiple cloning site (e.g., between BamHI and XhoI cleavage sites) of vector pYES 2. In another embodiment of the present invention, the recombinant vector may be a recombinant expression vector pBI121-GhGPAT obtained by inserting the GhGPAT gene into the multiple cloning site (e.g., between BamHI and SacI cleavage sites) of vector pBI 121.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. The yeast may specifically be Saccharomyces cerevisiae (e.g.Saccharomyces cerevisiae INVSC1) and the bacteria may be Agrobacterium (e.g.Agrobacterium tumefaciens GV 3101). In one embodiment of the present invention, the recombinant microorganism may be the recombinant yeast INVSC1(pYES2-GhGPAT) carrying the recombinant expression vector pYES 2-GhGPAT. In another embodiment of the invention, the recombinant microorganism is recombinant Agrobacterium pBI121-GhGPAT/GV3101 carrying recombinant expression vector pBI 121-GhGPAT.

In the application, the purpose of plant breeding is to cultivate a new crop variety with high oil content in seeds.

The second object of the present invention is to provide a method for constructing a recombinant yeast having an improved triglyceride production.

The construction method of the recombinant yeast with the improved triglyceride yield, provided by the invention, comprises the following steps: the coding gene of the GhGPAT protein is introduced into a receptor yeast to obtain the recombinant yeast with improved triglyceride yield.

Furthermore, the coding gene of the GhGPAT protein is introduced into the receptor yeast through a recombinant expression vector. The recombinant expression vector is obtained by inserting the coding gene of GhGPAT protein into an expression vector.

Furthermore, the nucleotide sequence of the coding gene of the GhGPAT protein is shown as a sequence 1.

The expression vector may be a pYES2 vector. The recombinant expression vector can be a recombinant expression vector pYES2-GhGPAT obtained by inserting the GhGPAT gene into a multiple cloning site (such as between BamHI and XhoI enzyme cutting sites) of a pYES2 vector.

The recipient yeast can be Saccharomyces cerevisiae; the Saccharomyces cerevisiae may be Saccharomyces cerevisiae INVScI.

The recombinant yeast with improved triglyceride yield constructed according to the method and the application thereof in preparing triglyceride also belong to the protection scope of the invention.

It is a third object of the present invention to provide a process for preparing triglycerides.

The method for preparing triglyceride provided by the invention comprises the step of carrying out induction culture on the recombinant yeast.

Further, the induction culture comprises the following steps: activating the recombinant yeast in an SC-ura amplification liquid culture medium, centrifuging when OD600 is 2.6, and collecting precipitates; then suspending the precipitate by SD-ura induced liquid medium to make OD600 of the culture system be 1.3, carrying out induced culture on a shaking table, centrifuging, and collecting thallus precipitate; the bacterial pellet contains triglyceride.

Further, the induction culture may be specifically performed according to the following steps: the recombinant yeast is activated in 30mL of SC-ura amplification liquid culture medium, when OD600 is 2.6, centrifugation is carried out at room temperature and 5000rpm for 5min, precipitates are collected, 60mL of SD-ura induction liquid culture medium (containing 2% of galactose) is used for resuspending the precipitates to enable the OD600 of a culture system to be 1.3, then the culture system is placed on a shaking table at 30 ℃ for induction culture at 180rpm/min, centrifugation is carried out at 5000rpm for 5min after 24h of culture, and thallus precipitates are collected.

It is a final object of the present invention to provide a method for breeding a transgenic plant with improved oil content.

The method for cultivating the transgenic plant with the improved oil content comprises the steps of improving the content and/or activity of GhGPAT protein in a receptor plant to obtain the transgenic plant; the transgenic plant has a higher oil content than the recipient plant.

Further, the oil content is seed oil content.

The method for improving the content and/or activity of the GhGPAT protein in the receptor plant is to over-express the GhGPAT protein in the receptor plant. The overexpression method is to introduce a GhGPAT protein coding gene into a receptor plant.

Furthermore, the coding gene of the GhGPAT protein is introduced into a receptor plant through a recombinant expression vector.

The recombinant expression vector is obtained by inserting the coding gene of GhGPAT protein into an expression vector.

The nucleotide sequence of the coding gene of the GhGPAT protein is shown as a sequence 1.

The expression vector may be a pBI121 vector. The recombinant expression vector can be pBI121-GhGPAT obtained by inserting the GhGPAT gene into a multiple cloning site (such as between BamHI and SacI enzyme cutting sites) of a pBI121 vector.

The expression vector carrying the coding gene of the GhGPAT protein can transform plant cells or tissues by using conventional biological methods such as direct DNA transformation, conductance, agrobacterium mediation and the like, and the transformed plant cells or tissues are cultivated into plants.

In the above application or method, the plant may be a monocotyledon or a dicotyledon. Further, the dicotyledonous plant can be cotton and arabidopsis. Further, the arabidopsis thaliana may specifically be colombia ecotype (Col).

The invention provides GhGPAT protein related to oil content of seeds in cotton and a coding gene thereof, and has important theoretical and practical significance for further clarifying molecular mechanism of oil content of plant seeds and cultivating new crop varieties with high oil content of seeds by genetic engineering technology and means.

Drawings

FIG. 1 is an electropherogram of PCR amplification of the GhGPAT gene. Note: m is DNA molecular marker MarkerIII, 1 is electrophoresis result after GhGPAT gene amplification.

FIG. 2 is a schematic diagram of the structure of a yeast expression vector for the GhGPAT gene.

FIG. 3 is an electrophoretogram of PCR product of recombinant yeast INVSC1(pYES 2-GhGPAT). Note: m is DNA molecular marker MarkerIII, and 1-7 is recombinant yeast INVSC1(pYES2-GhGPAT) PCR product.

FIG. 4 shows the relative expression level of GhGPAT in transgenic yeast. Note: CK 1: a control yeast; CK 2: recipient yeast INVScI; OE-1, OE-2, OE-3 and OE-4 are recombinant yeast strains.

FIG. 5 shows the triglyceride content of the transgenic yeast. Note: CK 1: a control yeast; CK 2: recipient yeast INVScI; OE-1, OE-2, OE-3 and OE-4 are recombinant yeast strains.

FIG. 6 is a schematic diagram of the structure of a plant expression vector for the GhGPAT gene.

FIG. 7 is the PCR identification electrophoresis of transgenic plants. Note: 1 is a wild type arabidopsis plant, 2 is a transgenic empty load positive arabidopsis plant, 3-8 is a transgenic GhGPAT gene positive arabidopsis plant, and M is a DNA molecular marker MarkerIII.

FIG. 8 shows the expression level of the GhGPAT gene in wild type Arabidopsis thaliana and three GhGPAT gene-transferred T3-generation homozygous lines. Note: WT is wild type Arabidopsis thaliana; CK is an Arabidopsis T3 generation strain of unloaded pBI 121; OE-1, OE-4 and OE-14 are GhGPAT-transferred Arabidopsis thaliana T3 generation strains, respectively.

FIG. 9 shows the results of the determination of the total oil content in seeds of wild type Arabidopsis thaliana and three GhGPAT gene-transferred T3 generation homozygous lines. Note: WT is wild type Arabidopsis thaliana; CK is an Arabidopsis T3 generation strain of unloaded pBI 121; OE-1, OE-4 and OE-14 are GhGPAT-transferred Arabidopsis thaliana T3 generation strains, respectively. Indicates that there is significant difference between wild type arabidopsis and transgenic lines.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The cotton high oil material 3008 in the following examples is described in the literature: zang X, Geng X, Ma L, et al.A genome-wide analysis of the phospholipid, diacylglycerol acyl transferase gene family in Gossypium [ J ]. BMC genetics, 2019,20(1):402. in; the biological material is available to the applicant only for the purpose of repeating the relevant experiments of the present invention and is not available for other uses.

The yeast expression vector pYES2 in the examples below is a product of Youbao organisms, cat # VT 1351.

Saccharomyces cerevisiae (INVScI) in the following examples is a product of Shanghai Toshidi Biotech, Inc. under the trade designation YC 1050.

pBI121 in the following examples is described in the literature: chen, P.Y., Wang, C.K., Song, S.C., & To, K.Y. (2003). Complete sequence of the binary vector pBI121 and its application in a closed T-DNA insertion from genetic plants, molecular weaving, 11(4), 287-293; the biological material is available to the applicant only for the purpose of repeating the relevant experiments of the present invention and is not available for other uses.

The SC-ura amplification liquid medium formulations in the following examples are as follows: weighing 8g of SD-ura (yeast defective culture medium SD-ura is a product of Beijing Sorleibao science and technology Co., Ltd., product number S0620) into 900mL of pure water, adjusting pH to 5.8, autoclaving at 121 deg.C for 15min, and adding 100mL of glucose solution with 20% concentration after sterilization when the temperature is reduced to about 55 deg.C.

The formulation of SD-ura induction broth (containing 2% galactose) in the following examples is as follows: SD-ura8g was weighed into 900mL of purified water, the pH was adjusted to 5.8, autoclaving was carried out at 121 ℃ for 15min, and 100mL of a 20% sterilized galactose solution and 100mL of a 10% sterilized raffinose solution were added after the temperature was reduced to about 55 ℃.

Example 1 cloning of Cotton GhGPAT Gene

1. Obtaining of cDNA

Extracting the total RNA of 5DPA-30DPA immature embryo mixed sample after cotton high oil material 3008 blooms, and performing reverse transcription to obtain cDNA.

2. PCR amplification

And (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and GhGF and GhGR as primers to obtain a PCR amplification product with the size of 1503 bp. The primer sequences are as follows:

primer GhF: 5'-ATGGGGGCTTACCGTTGT-3', respectively;

primer GhR: 5'-TTAAAGTACTAGAGCCTTTTCCTTT-3' are provided.

Reaction system for PCR amplification described above (total volume 50. mu.l): mu.l of cDNA, 1.5. mu.l each of GhF primer and GhR primer (10. mu.M), 5. mu.l of 10 XPCR Buffer for KOD-PLUS-Neo, 5. mu.l of dNTP (2mM each), MgSO₄(25mM each) 3. mu.l, KOD-PLUS-Neo enzyme 1. mu.l, ddH₂O 30μl。

Reaction procedure for the above PCR amplification: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 57 ℃ for 30s, extension at 68 ℃ for 45s, and 32 cycles; extension at 68 ℃ for 10 min.

3. PCR amplification product detection

The PCR amplification product obtained in step 2 was detected by 0.8% agarose gel electrophoresis, and the electrophoresis result showed a target band of about 1.5kb (FIG. 1), and gel recovery was carried out by a gel recovery kit to obtain a band fragment of about 1.5kb, which was ligated to pMD18-T (TAKARA, D101A), and transformed into E.coli competent DH 5. alpha. and positive clones were screened by the blue-white screening method and sequenced.

Sequencing results show that the positive cloning vector (named as pMD18-T-GhGPAT) is a vector pMD18-T in which the nucleotide shown in the sequence 1 in the sequence table is inserted, the gene shown by the nucleotide is GhGPAT, the coding region is from the 1 st to the 1503 th position, the protein coded by the gene is named as GhGPAT, the protein contains 500 amino acids, and the sequence 3 in the sequence table is the amino acid sequence of the protein.

Example 2 Effect of heterologous overexpression of the GhGPAT Gene on triglyceride content in Saccharomyces cerevisiae

Construction of recombinant Yeast

1. Obtaining of target Gene GhGPAT

The PCR amplification product is obtained by using pMD18-T-GhGPAT in the embodiment 1 as a template and adopting GhYF and GhYR primers for PCR amplification.

Primer GhYR: 5' -taccgagctcggatccATGGGGGCTTACCGTTGT-3' (the underlined sequence contains the recognition sequence for the restriction enzyme BamHI and the fragment on the vector);

primer GhYR: 5' -tagatgcatgctcgagTTAAAGTACTAGAGCCTTTTCCTTT-3' (the sequence shown underlined contains the recognition sequence for the restriction enzyme XhoI and the fragment on the vector).

2. Construction of recombinant Yeast expression vector pYES2-GhGPAT

1) The yeast expression vector pYES2 was digested with restriction enzymes BamHI and XhoI and the vector backbone was recovered.

2) Recovering and purifying the PCR amplification product containing the enzyme cutting sites obtained in the step 1.

3) Connecting the vector skeleton obtained in the step 1) with the recovered product obtained in the step 2) to form a recombinant yeast expression vector pYES2-GhGPAT (figure 2).

4) Transforming the ligation product in the step 3 into escherichia coli DH5 alpha by heat shock, placing the escherichia coli DH5 alpha in an incubator at 37 ℃ for inversion overnight culture, and selecting positive clones for sequencing; the sequencing result was correctly named pYES2-GhGPAT/DH5 alpha.

3. Construction and identification of recombinant yeast INVSC1(pYES2-GhGPAT)

1) Construction of recombinant Yeast INVSC1(pYES2-GhGPAT)

And (3) transforming the recombinant yeast expression vector pYES2-GhGPAT constructed in the step (2) into Saccharomyces cerevisiae (INVScI) by adopting a LiAc method, and screening to obtain the INVScI (pYES 2-GhGPAT). Meanwhile, the yeast expression vector pYES2 is transformed into Saccharomyces cerevisiae (INVScI) according to the method, and the yeast INVScI (pYES2) is obtained by screening.

2) Identification of recombinant Yeast INVSC1(pYES2-GhGPAT)

Selecting a single colony of a recombinant, activating the single colony in an SD-ura liquid culture medium, carrying out PCR amplification verification on an INVscI (pYES2-GhGPAT) single colony bacterial liquid and an INVscI (pYES2) single colony bacterial liquid by using GhYF and GhYR primers, and carrying out PCR amplification to obtain a recombinant yeast INVscI (pYES2-GhGPAT) with a target band of 1503bp, namely a positive recombinant yeast INVSC1(pYES2-GhGPAT), namely a positive transgenic GhGPAT yeast.

The identification result shows that: the PCR amplification of recombinant yeast INVscI (pYES2-GhGPAT) resulted in 1503bp of the target band, whereas the transfer empty vector yeast INVscI (pYES2) did not have the target band. The electrophoresis results of the PCR products of the partially positive recombinant yeast INVscI (pYES2-GhGPAT) are shown in FIG. 3, and they were named recombinant yeast INVscI (pYES2-GhGPAT) OE 1-recombinant yeast INVscI (pYES2-GhGPAT) OE7, respectively. Positive recombinant yeasts INVScI (pYES2-GhGPAT) OE1, OE2, OE3, OE4 were selected for the following experimental analysis.

Second, induced expression, expression quantity analysis and triglyceride content determination of recombinant yeast

1. Inducible expression of recombinant yeast

Respectively activating correctly identified positive recombinant yeast INVscI (pYES2-GhGPAT), empty vector yeast INVscI (pYES2) (CK1) and wild-type yeast INVscI (CK2) in 30mL of SC-ura amplification liquid culture medium, centrifuging at room temperature of 5000rpm for 5min when OD600 is 2.6, removing supernatant and collecting precipitate; the pellet was resuspended in 60mL of SD-ura induction liquid medium (containing 2% galactose) so that OD600 of the culture systems of the recombinant yeast strain, the empty vector-transferred yeast strain and the wild-type yeast strain were all 1.3, and then the resultant was placed on a 30 ℃ shaker at 180rpm/min overnight (24 hours) to induce expression of the protein, followed by centrifugation at 5000rpm for 5 minutes, and the pellet was collected.

2. Analysis of expression level

RNA of each yeast precipitate obtained in the step 1 is extracted by a yeast RNA extraction kit (R6870-01, Zhengzhou Qianji commercial Co., Ltd.), cDNA is reversely transcribed, the obtained cDNA is diluted by 3 times and used as a template of qRT-PCR, primers Gh9qF and Gh9qR are adopted, and qRT-PCR is carried out by taking 18S as an internal reference gene.

Gh9F：5’-ATCGAATCAGACAGTGGCTGCG-3’；

Gh9R：5’-CCGACCCCGAAACAAGCTCAAT-3’；

18SF：5’-TTAGTTGGTGGAGTGATTTG-3’；

18SR：5’-GGTGGCTCTGTCAGTGTAG-3’。

The results are shown in FIG. 4 and show that: compared with the empty vector yeast (CK1) and the wild-type yeast (CK2), the expression level of the GhGPAT gene in the 4 positive recombinant yeasts is remarkably increased, and the expression level of the GhGPAT gene is not detected in the empty vector yeast (CK1) and the wild-type yeast (CK 2).

3. Measurement of triglyceride content

The water content in each yeast pellet obtained in step 1 was dried by vacuum pumping at low temperature, and the triglyceride content in each yeast pellet was measured according to a yeast fat measurement kit (E1013, beijing prilley gene technology ltd).

The results are shown in FIG. 5 and show that: the triglyceride content in the bacterial precipitates of the 4 positive recombinant yeasts OE-1, OE-2, OE-3 and OE-4 is 1683.7 mu mol/L, 2407.8 mu mol/L, 1822.8 mu mol/L and 2467.8 mu mol/L respectively, and the triglyceride content in the bacterial precipitates of the empty vector yeast (CK1) and the wild type yeast (CK2) is 1359.5 mu mol/L and 1265.5 mu mol/L respectively. Compared with empty vector-transferred yeast (CK1) and wild-type yeast (CK2), the triglyceride (CK1) content of 4 positive recombinant yeasts OE-1, OE-2, OE-3 and OE-4 is obviously improved to reach an obvious level, and the triglyceride content of the empty vector-transferred yeast (CK1) and the wild-type yeast (CK2) is not obviously different. The GhGPAT has the function of improving the content of the triglyceride of the yeast.

Example 3 acquisition and phenotypic analysis of GhGPAT-transgenic Arabidopsis thaliana

Construction of plant expression vector pBI121-GhGPAT

1. The plant binary expression vector pBI121 was digested with restriction enzymes BamHI and SacI, and the vector backbone was recovered.

2. PCR amplification was performed using pMD18-T-GhGPAT from example 1 as a template and Gh9F1 and Gh9R1 primers to obtain a GhGPAT gene PCR product. And (3) carrying out 0.8% agarose gel electrophoresis detection on the GhGPAT gene PCR product. Recovering and purifying the GhGPAT gene PCR product containing the enzyme cutting sites.

Primer GhF 1: 5' -ggactctagaggatccATGGGGGCTTACCGTTGT-3' (the sequence underlined contains the recognition sequence for the restriction enzyme BamHI and the fragment on the vector);

primer GhR 1: 5' -gatcggggaaattcgagctcTTAAAGTACTAGAGCCTTTTCCTTT-3' (the sequence shown underlined contains the recognition sequence for the restriction enzyme SacI and the fragment on the vector).

3. And (2) connecting the vector skeleton obtained by enzyme digestion in the step (1) and the GhGPAT gene PCR product recovered in the step (2) by an infusion method to obtain a plant expression vector pBI121-GhGPAT (figure 6).

4. Transforming the ligation product in the step 3 into escherichia coli DH5 alpha by a heat shock method, inverting at 37 ℃ for overnight culture, and selecting positive clones for sequencing; the sequencing result was correctly named pBI121-GhGPAT/DH5 alpha.

Second, construction of recombinant Agrobacterium pBI121-GhGPAT/GV3101

Transferring the plant expression vector pBI121-GhGPAT obtained in the first step into competent cells of Agrobacterium tumefaciens GV3101 (purchased from Zheng Zhongshang physician science and technology Co., Ltd.) by freeze thawing, and culturing at 28 ℃ for 2 days in LB solid culture dish containing 50. mu.g/ml kanamycin and 50. mu.g/ml rifampicin; positive monoclonals were identified by colony PCR (using primers GhF1 and GhR1 in example 3) and the correct recombinant Agrobacterium was named pBI121-GhGPAT/GV 3101.

Meanwhile, pBI121 is transferred into competent cells of Agrobacterium tumefaciens GV3101 according to the method to obtain recombinant Agrobacterium pBI121/GV 3101.

Obtaining and identifying GhGPAT-converted Arabidopsis thaliana

1. Culturing recombinant Agrobacterium pBI121-GhGPAT/GV3101 and pBI121/GV3101 on a shaker at 28 deg.C until OD600 is 1.6-2.0; the cells were collected by centrifugation and resuspended in MS solution (macroelements, 5% sucrose, 40. mu.l/500 ml Silwet L-77) to make Agrobacterium-infected solution with OD600 of 0.8-1.0.

2. Respectively infecting Columbia ecotype arabidopsis thaliana (Col-0) (seeds are purchased from Salk Institute Genomic Analysis Laboratory) with the agrobacterium infection solution prepared in the step 1 by a flower dipping method, harvesting the seeds after cultivation, paving the seeds on an MS culture medium containing 50 mu g/ml kanamycin, and transferring T1 transgenic plants to be screened to culture soil for normal growth when the transgenic plants grow to about 4 leaves so as to respectively obtain T1 generation GhGPAT transgenic arabidopsis thaliana and empty vector transfer arabidopsis thaliana.

3. Extracting genome DNA of leaves of T1-generation GhGPAT arabidopsis single plants and empty vector arabidopsis single plants, and performing PCR amplification by using the genome DNA as a template and using kanF and kanR primers to identify positive plants; meanwhile, wild arabidopsis thaliana is used as a negative control, and a plant expression vector pBI121-GhGPAT is used as a positive control.

KanF：5’-ATGATTGAACAAGATGGATTG-3’；

KanR：5’-TCAGAAGAACTCGTCAAGAAG-3’。

The results are shown in FIG. 7 and show that: the transgenic GhGPAT Arabidopsis plant, the transgenic empty vector Arabidopsis plant and the positive control plasmid are amplified to form a 500bp strip, and the wild type Arabidopsis (negative control) is not amplified to form the strip.

4. And (3) screening seeds of GhGPAT Arabidopsis thaliana transformed from T1 generations by using the same kanamycin MS culture medium, observing the separation condition of plants of T2 generations, and finally obtaining 8 strains of GhGPAT Arabidopsis thaliana transformed from T2 generations. 3T 2 generation GhGPAT Arabidopsis thaliana strains (OE-1, OE-4 and OE-14) are randomly selected to propagate to T3 generation for expression quantity analysis. The method comprises the following specific steps:

extracting RNA of 3T 3-generation GhGPAT Arabidopsis strains, and performing qRT-PCR identification by using cDNA obtained by reverse transcription as a template and the GhqF and GhqR primers, wherein AtActinF and AtActinR are used as reference genes, and wild Arabidopsis is used as a reference.

AtActinF：5’-TGGCATCATACTTTCTACAA-3’；

AtActinR：5’-CCACCACTGAGCACAATGTT-3’。

The results are shown in fig. 8 and show that: the GhGPAT genes in 3T 3-generation GhGPAT Arabidopsis thaliana strains (OE-1, OE-4 and OE-14) have expression levels, but the expression levels of the GhGPAT genes cannot be detected in unloaded Arabidopsis thaliana (CK) and wild Arabidopsis thaliana (WT).

Determination of oil content of GhGPAT-converted Arabidopsis thaliana seed

The seed oil content (%) of wild type Arabidopsis thaliana, empty vector transfer Arabidopsis thaliana and T3 generation GhGPAT Arabidopsis thaliana strains OE-1, OE-4 and OE-14 is determined by a nuclear magnetic resonance imaging analyzer (NMI 20-analysis). The specific measurement procedure was performed according to the instructions.

The results are shown in fig. 9 and indicate that: the seed oil content of T3 generation GhGPAT Arabidopsis thaliana strains OE-1, OE-4 and OE-14 is 21.745%, 22.332% and 22.327%, respectively, and the seed oil content of wild type Arabidopsis thaliana (WT) is 18.601%. Compared with wild Arabidopsis (WT), the seed oil content of GhGPAT transgenic Arabidopsis strains (OE-1, OE-4 and OE-14) is increased. The oil content of the empty vector Arabidopsis thaliana (CK, 17.545%) is not obviously different from that of the wild Arabidopsis thaliana (WT). The GhGPAT has the function of improving the content of the vegetable oil.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

<120> protein related to plant oil content increase, coding gene and application thereof

<160>3

<170>PatentIn version 3.5

<210>1

<211>1503

<212>DNA

<213>Artificial Sequence

<400>1

atgggggctt accgttgttt cgaaccgatt tccaaatgta gcagcgacgg aagatcgaat 60

cagacagtgg ctgcggattt agatgggacc cttctcgtgt cacgaagtgc ttttccttat 120

ttcatgcttg ttgctcttga agctggtagc cttgttagag cacttgttct gttaacatcc 180

gttccatttg tgtactttac gtacttgttc atctctgagt ccgtagccat taataccttc 240

attttcatca ccttctccgg gctaaaagtt agagacattg agcttgtttc ggggtcggtc 300

ttgcccaagt tttatgccga ggatgttcat ccagacacct ggagagtttt cagctccttt 360

ggcaaacgat acattattac agcgagtcct aggatcatgg tggaaccctt tgccaagaca 420

tatttaggag ctgacaaggt tattggaaca gagttagaag tgacgaaatc tggcagggca 480

actggttttg ccattaaacc tggagttctc gtcggagaac acaaaagggc tgccgttttg 540

aaagaattcg gcaccaactt gccagatttg gggctaggag acagagaaac cgaccatgat 600

ttcatgtcat tatgcaagga aggatacatg gtgccaagaa ccaaatgcga accattgcca 660

aggaacaagc ttctaagcca tgttatattt catgagggcc gcctagtcca aaggccaacc 720

cctttggctg ctctgttgac cttcctatgg cttcctatag gcttcatcct ctccctactc 780

agggtctaca ccaacatccc gttaccggag cgaattgctc gatacaatta caggttgctg 840

gggatcaaat tgattgttaa gggtacccct ccaccagcac ctaggaaagg ccaaagtgga 900

gtcctctttg tttgcaacca tagaacagtt ttagacccag ttgtcacagc ggttgcctta 960

ggccgaaaaa tcagttgtgt tacttacagt attagcaaat tcactgaaat tatttcgcct 1020

attcgggccg ttgcattatc gagagaaaga gaaaaagatg cagccgacat caagcgcctt 1080

ctcgaggaag gtgacttggt tatttgtcct gaagggacta cctgccgaga accttttctg 1140

ctaagattca gtgcactgtt cgctgaactc actgatagaa ttgtgccggt cgcaatcaac 1200

acaaaacaaa cagttttcca cgggacaaca gttcggggac acaaattgtt ggatccttat 1260

tttgtattca tgaatccgat gcctacttac gagatcacct tcttgaacca gctgcctaca 1320

gagcttactt gcaaaggtgg aaaatcagcc attgaagttg caaactacat ccaaagggtg 1380

ctggctggga cattgggatt tgagtgcacc aacttgacaa ggaaggacaa gtatgccatg 1440

cttgcaggaa cggacggtcg ggttccatcc aagaaagaaa aggaaaaggc tctagtactt 1500

taa 1503

<210>2

<211>2339

<212>DNA

<213>Artificial Sequence

<400>2

atgggggctt accgttgttt cgaaccgatt tccaaatgta gcagcgacgg aagatcgaat 60

cagacagtgg ctgcggattt agatgggacc cttctcgtgt cacgaagtgc ttttccttat 120

ttcatgcttg ttgctcttga agctggtagc cttgttagag cacttgttct gttaacatcc 180

gttccatttg tgtactttac gtacttgttc atctctgagt ccgtagccat taataccttc 240

attttcatca ccttctccgg gctaaaagtt agagacattg agcttgtttc ggggtcggtc 300

ttgcccaagt tttatgccga ggatgttcat ccagacacct ggagagtttt cagctccttt 360

ggcaaacgat acattattac agcgagtcct aggatcatgg tggaaccctt tgccaagaca 420

tatttaggag ctgacaaggt tattggaaca gagttagaag tgacgaaatc tggcagggca 480

actggttttg ccattaaacc tggagttctc gtcggagaac acaaaagggc tgccgttttg 540

aaagaattcg gcaccaactt gccagatttg gggctaggag acagagaaac cgaccatgat 600

ttcatgtcat tatgcaaggt tcagtctcac taactaaacc ctcgctcctt tctctttttt 660

actgccatat ttgaacaatt ttacaaatgg tttcttagta taggatctaa cacatattcc 720

ttaaacataa gattatatat gtttggtgag agcgtttatg atccaataat ggttttgctt 780

gagatgcctg tccctttaga tctttagatt gctattgcct ttttatagtt acaaaattca 840

tatttcaatg gtccacagtg ttccatactc ctaattttac gtttagcttt acccaacacc 900

tttcctaatc ctgaaagcct tttctcctct agctttttat tttttttatt ttttttattt 960

ttttgctgaa aagattatca aagaacaagt ggcaattcaa ttttcaagcc agtttttctt 1020

caattaattt tctactaaag aacacaactt tttcgtgcca taaccactct tttgtacctc 1080

tagtttttgt acaaatcatg gtcaatttgt tccaagcagc agcgccatcc acgtgatgtt 1140

ttcaatgggt tgtctcccaa ttaataataa gaagaaaagg aaaagtactt ttacatgaaa 1200

ctaacatgta tcataaatat agtactaata cttttgaatt aactctgtaa taataattaa 1260

cacaaaaaat taacatgtgc atggattcct tacgtataac ttcatattct tgagctgcaa 1320

tattatttgg agttaatgca tggcaagatg gttagatgat tgttggtgaa gtgaacacca 1380

aaaagtttca agttttattg aataaactta tggaattgat tggcattttt tacattgatt 1440

ttgtttatgt acaggaagga tacatggtgc caagaaccaa atgcgaacca ttgccaagga 1500

acaagcttct aagccatgtt atatttcatg agggccgcct agtccaaagg ccaacccctt 1560

tggctgctct gttgaccttc ctatggcttc ctataggctt catcctctcc ctactcaggg 1620

tctacaccaa catcccgttg ccggagcgaa ttgctcgata caattacagg ttgctgggga 1680

tcaaattgat tgttaagggt acccctccac cagcacctag gaaaggccaa agtggagtcc 1740

tctttgtttg caaccataga acagttttag acccagttgt cacagcggtt gccttaggcc 1800

gaaaaatcag ttgtgttact tacagtatta gcaaattcac tgaaattatt tcgcctattc 1860

gggccgttgc attatcgaga gaaagagaaa aagatgcagc cgacatcaag cgccttctcg 1920

aggaaggtga cttggttatt tgtcctgaag ggactacctg ccgagaacct tttctgctaa 1980

gattcagtgc actgttcgct gaactcactg atagaattgt gccggtcgca atcaacacaa 2040

aacaaacagt tttccacggg acaacagttc ggggacacaa attgttggat ccttattttg 2100

tattcatgaa tccgatgcct acttacgaga tcaccttctt gaaccagctg cctacagagc 2160

ttacttgcaa aggtggaaaa tcagccattg aagttgcaaa ctacatccaa agggtgctgg 2220

ctgggacatt gggatttgag tgcaccaact tgacaaggaa ggacaagtat gccatgcttg 2280

caggaacgga cggtcgggtt ccatccaaga aagaaaagga aaaggctcta gtactttaa 2339

<210>3

<211>500

<212>PRT

<213>Artificial Sequence

<400>3

Met Gly Ala Tyr Arg Cys Phe Glu Pro Ile Ser Lys Cys Ser Ser Asp

1 5 10 15

Gly Arg Ser Asn Gln Thr Val Ala Ala Asp Leu Asp Gly Thr Leu Leu

20 25 30

Val Ser Arg Ser Ala Phe Pro Tyr Phe Met Leu Val Ala Leu Glu Ala

35 40 45

Gly Ser Leu Val Arg Ala Leu Val Leu Leu Thr Ser Val Pro Phe Val

50 55 60

Tyr Phe Thr Tyr Leu Phe Ile Ser Glu Ser Val Ala Ile Asn Thr Phe

65 70 75 80

Ile Phe Ile Thr Phe Ser Gly Leu Lys Val Arg Asp Ile Glu Leu Val

85 90 95

Ser Gly Ser Val Leu Pro Lys Phe Tyr Ala Glu Asp Val His Pro Asp

100 105 110

Thr Trp Arg Val Phe Ser Ser Phe Gly Lys Arg Tyr Ile Ile Thr Ala

115 120 125

Ser Pro Arg Ile Met Val Glu Pro Phe Ala Lys Thr Tyr Leu Gly Ala

130 135 140

Asp Lys Val Ile Gly Thr Glu Leu Glu Val Thr Lys Ser Gly Arg Ala

145 150 155 160

Thr Gly Phe Ala Ile Lys Pro Gly Val Leu Val Gly Glu His Lys Arg

165 170 175

Ala Ala Val Leu Lys Glu Phe Gly Thr Asn Leu Pro Asp Leu Gly Leu

180 185 190

Gly Asp Arg Glu Thr Asp His Asp Phe Met Ser Leu Cys Lys Glu Gly

195 200 205

Tyr Met Val Pro Arg Thr Lys Cys Glu Pro Leu Pro Arg Asn Lys Leu

210 215 220

Leu Ser His Val Ile Phe His Glu Gly Arg Leu Val Gln Arg Pro Thr

225 230 235 240

Pro Leu Ala Ala Leu Leu Thr Phe Leu Trp Leu Pro Ile Gly Phe Ile

245 250 255

Leu Ser Leu Leu Arg Val Tyr Thr Asn Ile Pro Leu Pro Glu Arg Ile

260 265 270

Ala Arg Tyr Asn Tyr Arg Leu Leu Gly Ile Lys Leu Ile Val Lys Gly

275 280 285

Thr Pro Pro Pro Ala Pro Arg Lys Gly Gln Ser Gly Val Leu Phe Val

290 295 300

Cys Asn His Arg Thr Val Leu Asp Pro Val Val Thr Ala Val Ala Leu

305 310 315 320

Gly Arg Lys Ile Ser Cys Val Thr Tyr Ser Ile Ser Lys Phe Thr Glu

325 330 335

Ile Ile Ser Pro Ile Arg Ala Val Ala Leu Ser Arg Glu Arg Glu Lys

340 345 350

Asp Ala Ala Asp Ile Lys Arg Leu Leu Glu Glu Gly Asp Leu Val Ile

355 360 365

Cys Pro Glu Gly Thr Thr Cys Arg Glu Pro Phe Leu Leu Arg Phe Ser

370 375 380

Ala Leu Phe Ala Glu Leu Thr Asp Arg Ile Val Pro Val Ala Ile Asn

385 390 395 400

Thr Lys Gln Thr Val Phe His Gly Thr Thr Val Arg Gly His Lys Leu

405 410 415

Leu Asp Pro Tyr Phe Val Phe Met Asn Pro Met Pro Thr Tyr Glu Ile

420 425 430

Thr Phe Leu Asn Gln Leu Pro Thr Glu Leu Thr Cys Lys Gly Gly Lys

435 440 445

Ser Ala Ile Glu Val Ala Asn Tyr Ile Gln Arg Val Leu Ala Gly Thr

450 455 460

Leu Gly Phe Glu Cys Thr Asn Leu Thr Arg Lys Asp Lys Tyr Ala Met

465 470 475 480

Leu Ala Gly Thr Asp Gly Arg Val Pro Ser Lys Lys Glu Lys Glu Lys

485 490 495

Ala Leu Val Leu

500

Claims

Use of a GhGPAT protein in any one of the following m1) -m 5):

m1) regulating and controlling the content of triglyceride of yeast;

m2) regulating and controlling the content of vegetable oil;

m3) constructing a recombinant yeast with increased triglyceride production;

m4) cultivating transgenic plants with improved oil content;

m5) plant breeding;

the GhGPAT protein is a protein shown as a) or b) or c) or d) as follows:

a) the amino acid sequence is a protein shown in a sequence 3;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 3;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 3;

d) and (b) a protein having a homology of 75% or more than 75% with the amino acid sequence shown in the sequence 3 and having the same function.
2. Application of the biological material related to GhGPAT protein in any one of the following m1) -m 5):

m1) regulating and controlling the content of triglyceride of yeast;

m2) regulating and controlling the content of vegetable oil;

m3) constructing a recombinant yeast with increased triglyceride production;

m4) cultivating transgenic plants with improved oil content;

m5) plant breeding;

the biomaterial related to the GhGPAT protein is any one of the following A1) to A8):

A1) a nucleic acid molecule encoding a GhGPAT protein;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising the recombinant vector of a 4).
3. Use according to claim 2, characterized in that: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

1) the coding sequence is a cDNA molecule shown in a sequence 1 or a genome DNA molecule shown in a sequence 2;

2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the GhGPAT protein described in claim 1;

3) a cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the GhGPAT protein described in claim 1.
4. A construction method of recombinant yeast with improved triglyceride yield comprises the following steps: a recombinant yeast having an improved triglyceride production, which is obtained by introducing the gene encoding the GhGPAT protein according to claim 1 into a recipient yeast.
5. The method of claim 4, wherein: the coding gene of the GhGPAT protein is introduced into the receptor yeast through a recombinant expression vector;

or, the recombinant expression vector is a vector obtained by inserting the coding gene of the GhGPAT protein into an expression vector;

or, the nucleotide sequence of the coding gene of the GhGPAT protein is shown as a sequence 1;

or, the expression vector is pYES2 vector;

or the recipient yeast is saccharomyces cerevisiae.
6. A recombinant yeast having an increased production of triglycerides constructed according to the method of claim 4 or 5.
7. Use of the recombinant yeast of claim 6 for the preparation of triglycerides.
8. A method for producing triglycerides, comprising the step of subjecting the recombinant yeast of claim 6 to induction culture.
9. A method for producing a transgenic plant having an increased oil content, comprising the step of increasing the content and/or activity of GhGPAT protein according to claim 1 in a recipient plant to obtain a transgenic plant; the transgenic plant has a higher oil content than the recipient plant.
10. The method of claim 9, wherein: the method for increasing the content and/or activity of the GhGPAT protein in the receptor plant as claimed in claim 1 is to over-express the GhGPAT protein in the receptor plant;

or, the overexpression method is to introduce the coding gene of GhGPAT protein into a receptor plant;

or the nucleotide sequence of the coding gene of the GhGPAT protein is shown as a sequence 1.