CN112342235A - Application of GmDGAT2A in increasing soybean oil content and linoleic acid content - Google Patents

Abstract

The invention discloses an application of GmDGAT2A protein in improving soybean oil content and increasing linoleic acid content, wherein the amino acid sequence of the GmDGAT2A protein is shown as SEQ ID NO: 3, respectively. Experiments prove that the GmDGAT2A gene is specifically overexpressed in the cultivated soybean P03 seed, so that the oil content in the soybean seed can be increased, the content of linoleic acid in the oil can be specifically increased, and meanwhile, the overexpression of the GmDGAT2A gene does not influence other agronomic traits of the soybean. The invention meets the requirement of agricultural sustainable development, responds to the national policy of cultivating high-quality soybean varieties, and has important application value and market prospect in the aspects of breeding high-quality high-oil soybean new varieties, improving plant yield and quality, improving ecological environment and the like.

Description

Application of GmDGAT2A in increasing soybean oil content and linoleic acid content

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of GmDGAT2A in improving soybean oil content and increasing linoleic acid content.

Background

Soybeans are important oil plants and play an important role in human diet, livestock production, and industrial and medical industries. With the increasing world population and the increasing standard of living, the demand for soybeans, especially soybean oil, and beneficial fatty acids is increasing. In soybean oil, oleic acid and linoleic acid are the two largest fatty acids. The linoleic acid has the functions of reducing blood fat and softening blood vessels, can prevent or reduce the incidence rate of cardiovascular diseases, is particularly favorable for preventing and treating hypertension, hyperlipidemia, angina pectoris, coronary heart disease, atherosclerosis, senile obesity and the like, can prevent the deposition of serum cholesterol in blood vessel walls of human bodies, has the reputation of 'blood vessel scavenger', has the health-care effect of preventing and treating atherosclerosis and cardiovascular diseases, is fatty acid which can not be synthesized by human bodies, can be obtained only from diet and is necessary for the human bodies.

However, the available cultivated land area worldwide is decreasing year by year, and the planting area of soybeans is also decreasing. The cultivation of high oil and high linoleic soybean varieties is the most effective way to solve the contradiction between increasing demand and decreasing arable land area. The gene engineering means such as transgene and the like can more accurately realize the directional transfer of the known functional genes compared with the traditional breeding, and can regulate the expression of the transferred genes by selecting different promoters, and the transfer efficiency of the genes is very high, thereby providing the most direct and efficient method for the quality improvement of the soybeans with great difficulty. Therefore, the new soybean variety with high oil content and high linoleic acid content is bred by genetic engineering technology such as transgenosis and the like, and a wide application prospect is opened for soybean genetic breeding.

The GmDGAT2A gene (the gene number on the Phytozome website https:// phytozome.jgi. doe. gov/pz/port. html: Glyma09g32790) encodes the GmDGAT2A protein (diacylglycerol acyltransferase) which is involved in TAG biosynthesis and has a high degree of homology in plants, animals and algae. At present, the research on DGAT1 gene in plants is more, and the research finds that the oil content of seeds can be obviously reduced in the interference of the expression of rape DGAT1 and the arabidopsis DGAT1 mutant, and the catalytic substrate of DGAT1 is very wide. Relatively few studies have been conducted on DGAT 2. It was found in castor that over-expression of castor DGAT2(RcDGAT2) increased the hydroxy fatty acids in the seeds; DGAT2 from Brassica napus is more likely to catalyze the production of long chain polyunsaturated fatty acids such as linolenic acid. However, the function of GmDGAT2A gene in soybean is still very limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide the application of GmDGAT2A in improving the content of soybean oil and increasing the content of linoleic acid.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the invention firstly protects the application of GmDGAT2A protein: is S1) or S2):

s1) regulating fatty acid synthesis in plant tissues;

s2) cultivating transgenic plants with high oil and high linoleic acid;

in the above application, the GmDGAT2A protein can be a1) or a2) or a 3):

a1) the amino acid sequence is SEQ ID NO: 3;

a2) in SEQ ID No.: 3, the N end or/and the C end of the protein shown in the formula 3 is connected with a label to obtain a fusion protein;

a3) converting SEQ ID NO: 3 through substitution and/or purpose or deletion and/or addition of one or more amino acid residues, and/or the protein is obtained and is related to the regulation and control of fatty acid synthesis in plant tissues.

Wherein, SEQ ID NO: 3 consists of 337 amino acid residues.

The invention also protects the application of the nucleic acid molecule for coding the GmDGAT2A protein, namely S1) or S2):

s1) regulating and controlling the synthesis of fatty acid in plant tissues;

s2) cultivating transgenic plants with high oil and high linoleic acid.

In the above application, the nucleic acid molecule may be a DNA molecule represented by b1) or b 2):

b1) the coding region is SEQ ID NO: 2;

b2) a DNA molecule which is hybridized with the nucleotide sequence limited by b1) under strict conditions and codes the GmDGAT2A 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.

Wherein, SEQ ID NO: 2 consists of 1014 nucleotides.

The invention also protects the use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing a nucleic acid molecule encoding the GmDGAT2A protein according to claim 1, selected from S1) or S2):

s1) regulating fatty acid synthesis in plant tissues;

s2) cultivating transgenic plants with high oil and high linoleic acid;

in the above application, the nucleic acid molecule is a DNA molecule shown as b1) or b 2):

b1) the coding region is SEQ ID NO: 2;

b2) a DNA molecule which is hybridized with the nucleotide sequence limited by b1) under strict conditions and codes the GmDGAT2A protein.

The nucleotide sequence of the GmDGAT2A protein 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 the GmDGAT2A protein isolated according to 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 the GmDGAT2A protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID NO: 3, or 80% or more, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the GmDGAT2A protein. 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.

In any of the above applications, the regulating the synthesis of fatty acids in plant tissue may be increasing the oil content and linoleic acid content in the plant or decreasing the oil content and linoleic acid content in the plant.

The plant tissue is a seed.

In any of the above applications, the plant may be c1) or c2) as follows: c1) a dicotyledonous plant; c2) a monocot plant; preferably, the dicotyledonous plant is a crucifer or a leguminous plant.

More preferably, the crucifer is arabidopsis thaliana; preferably, the Arabidopsis thaliana is a Columbia-0 subtype of wild type Arabidopsis thaliana.

More preferably, the leguminous plant is soybean.

The invention also provides a method for cultivating the transgenic plant with high oil content and high linoleic acid content, which comprises the following steps: improving the expression quantity and/or activity of the GmDGAT2A protein in the starting plant to obtain a transgenic plant; compared with the original plant, the neutron oil content and the linoleic acid content of the transgenic plant are improved.

In the method, the expression level and/or activity of the GmDGAT2A protein in the starting plant can be improved by changing the promoter, so that the effect of improving the expression level and/or activity of any one of the GmDGAT2A proteins in the starting plant can be achieved.

The invention provides a seed specific promoter ole1 separated from soybean, which is used for starting the specific expression of GmDGAT2A in the seeds of transgenic materials and improving the expression quantity and activity of the GmDGAT2A under the load dead weight. The nucleotide sequence is shown as SEQ ID NO: 1, the preparation method is as follows.

In the method, the improvement of the expression level and/or activity of the GmDGAT2A protein in the starting plant is achieved by introducing a nucleic acid molecule encoding the GmDGAT2A protein into the starting plant.

In the method, the introduction of the nucleic acid molecule encoding the GmDGAT2A protein into the starting plant can be realized by introducing a recombinant vector into the starting plant; the recombinant vector is obtained by inserting a nucleic acid molecule encoding the GmDGAT2A protein into an expression vector.

The recombinant vector can be specifically a recombinant plasmid proole 1 GmDGAT2A-pBA 002. The recombinant plasmid pro ole1 GmDGAT2A-pBA002 can specifically replace a 35s promoter between recognition sequences of restriction enzymes NcoI and XbaI of a pBA002 vector with SEQ ID NO: 1, and the obtained recombinant plasmid proole 1-pBA 002. The small fragment between the XbaI and XhoI recognition sequences was then replaced with SEQ ID NO: 2, and the obtained recombinant plasmid proole 1, namely GmDGAT2A-pBA 002.

The transgenic plants may in particular be the OE1 and OE2 mentioned in the examples. In this case, the starting plant is soybean, specifically, cultivated soybean P03.

The invention also protects a plant breeding method, and the expression level of the GmDGAT2A in the plant is improved, so that the content of the vegetable oil is improved, and the content of the linoleic acid component is increased.

Advantageous effects

Experiments prove that the oil content of the soybean can be improved and the linoleic acid content in the oil can be increased by over-expressing the GmDGAT2A gene in the cultivated soybean P03 seeds; meanwhile, the oil body protein promoter is used for starting the GmDGAT2A to be over-expressed in seeds, and the agronomic characters such as the plant height, the branch number, the single plant pod number and the like of the transgenic soybean plant are not influenced. The invention meets the requirement of agricultural sustainable development, and has important application value and market prospect in breeding new soybean varieties with high oil and high linoleic acid, improving plant yield and quality, improving ecological environment and the like.

Drawings

FIG. 1 shows the functional verification of soybean oil body protein gene and the seed specific promoter used, wherein FIG. 1A shows the analysis of the expression level of soybean oil body protein gene in various tissues of soybean; FIG. 1B is a functional validation of a bean-specific promoter used for soybean transgenesis;

FIG. 2 is a map of the vector used for the soybean transgene;

FIG. 3 is the result of identification of transgenic soybean material, wherein FIG. 3A is the electrophoretogram of transgenic soybean material, and FIG. 3B is the analysis of expression level of soybean GmDGAT2A in transgenic soybean material;

FIG. 4 shows the measurement results of oil content in transgenic soybean seeds;

FIG. 5 is a transgenic soybean seed oil composition change analysis;

the specific implementation mode is as follows:

the experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Arabidopsis thaliana wild type Col-0 ecotype (Arabidopsis thaliana ecotype Columbia-0) seeds were purchased from the Arabidopsis thaliana biological research center (ABRC, https:// www.arabidopsis.org /). Hereinafter, the Arabidopsis thaliana wild type Col-0 ecotype seed is referred to as a wild type Arabidopsis thaliana seed, and the Arabidopsis thaliana wild type Col-0 ecotype is referred to as a wild type Arabidopsis thaliana.

The cultivated soybean P03 seed is from Jilin farm institute. Hereinafter, the soybean cultivar type P03 seed is simply referred to as cultivar type soybean seed, and the cultivar type soybean P03 is simply referred to as cultivar type soybean.

Agrobacterium tumefaciens GV3101 is described in: r. Berres, L. otten, B.Tinland et

al.Transformation of vitis tissue by different strains of Agrobacterium

tumefaciens containing the T_6b gene.Plant Cell Reports，1992(11):192-195.

Escherichia coli DH5 alpha (DE3) is a product of TAKARA-Baori physicians & Technics Co., Ltd.

The GmDGAT2A gene involved in the examples below was derived from soybean (Glycine max (Linn.) Merr.) and its amino acid sequence in the soybean genome is shown in SEQ ID NO: 3, the CDS sequence is shown as SEQ ID NO: 2, respectively. The seed-specific promoter gene sequence is derived from soybean, and the DNA sequence is shown as SEQ ID NO: 1 is shown.

Example 1 obtaining and identification of Soybean seed specific promoters

Identification of soybean oil body protein gene expression level

1. Extracting RNA from soybean root, stem, young leaf, old leaf, flower, immature seed, mature seed, young pod and old pod

Taking a proper amount of the soybean tissue sample, fully grinding, and continuously adding liquid nitrogen during the grinding until the sample is powdery; transferring the sample to 1.5mL tube containing an appropriate amount of RNAasso Plus, standing at room temperature for 5 minutes, centrifuging at 12000g 4 ℃ for 5 minutes, transferring the supernatant to a new 1.5mL tube, adding a volume of 1/5 RNAasso Plus of chloroform, shaking for homogenization, standing at room temperature for 5 minutes, centrifuging at 12000g 4 ℃ for 15 minutes, transferring the supernatant to a new 1.5mL tube, adding a volume of 0.5-1 times the volume of RNAasso Plus of isopropanol, standing at room temperature for 10 minutes, centrifuging at 12000g 4 ℃ for 10 minutes, washing the precipitate with 75% ethanol of the same volume as RNAasso Plus, centrifuging at 7500g 4 ℃ for 5 minutes, discarding the supernatant, retaining the precipitate, drying (not heat drying), dissolving in an appropriate amount of DEPC treated water (see the RNA extraction flowsheet in TAKARA RNAasso Plus manual). After the RNA precipitate is completely dissolved, the RNA concentration and quality of the sample are detected by electrophoresis and an ultraviolet spectrophotometer (A260/A280), and the sample is stored at-80 ℃.

2. Reverse transcription

2. mu.g of each of the RNA samples extracted above was inverted.

1.) Synthesis of cDNA

The following mixture was added to the reaction PCR tube:

quench on ice at 65 ℃ after 5min, add the following reverse transcription mix:

reacting at 30 deg.C for 10min, and reacting at 42 deg.C for 30 min; then after inactivation treatment at 95 ℃, the mixture is placed on ice for the next PCR amplification.

3. Designing a semi-quantitative PCR amplification primer and carrying out semi-quantitative RT-PCR:

the following primers were designed based on the CDS sequence of the oil body protein gene:

Forward：5'-TACTCCACTCAGGTCGTCAA-3'(SEQ ID NO：4)；

Reverse：5'-TGTAGATCCACGCCAGCACC-3'(SEQ ID NO：5)；

performing semi-quantitative PCR reaction on the product obtained by reverse transcription, and taking a cons6 gene which is highly conserved in soybean and is expressed in a constitutive mode as an internal reference, wherein the PCR reaction system (25 mu L system): ddH₂O, 9.5 μ L; mix, 12.5 μ L; rTaq enzyme, 0.5. mu.L; template DNA, 0.5. mu.L; sense Primer, 1 μ L; anti-sense Primer, 1. mu.L. The PCR reaction condition is pre-denaturation at 94 ℃ for 3 min; 30S at 94 ℃, 30S at 72 ℃ and 30S for 28 cycles; finally, extension is carried out for 10min at 72 ℃. And analyzing the expression quantity by using related software, analyzing the size of an amplified fragment of the amplification product of the oleosin gene by agarose gel electrophoresis according to an Ethidium Bromide (EB) staining signal, and taking a picture of the gel.

The results are shown in FIG. 1.A, and the semi-quantitative results show that the oil body protein gene is highly expressed only in immature seeds.

II, obtaining and functional identification of oil body protein gene promoter

1. Cloning of the promoter of the oil body protein Gene

Extracting total DNA of soybean (variety is Glycine max Wm82.a2.v1) by using an SDS method, designing a primer to amplify a target promoter sequence, adding SalI and BamHI enzyme cutting sites, and amplifying the primers as follows:

Forward：5’-GTCGACTTCTATTCAGGAGGTGGTTG-3’(SEQ ID NO：6)；

Reverse：5’-GGATCCGAGTTTTGAGTGAAGAGTGAG-3’(SEQ ID NO：7)；

the amplified sequence size is 1601 bp. The cloned product is connected with a T vector (purchased from PROMEGA), a single colony is selected for shake bacteria, plasmid is extracted for sequencing, and the correct sequence of the promoter is verified. The plasmid and pBI101 vector (purchased from Biovector Science Lab) which are verified to be correct in sequencing are subjected to double digestion by SalI and BamHI restriction enzymes, the target fragment and the vector fragment which are recovered by digestion are connected at 4 ℃ overnight, the connection product is transferred into escherichia coli competent cells, and the cells are plated for overnight culture after being recovered at 37 ℃ for 1 h. And (4) selecting a single colony quality-improving particle for sequencing, and verifying that the sequence of the target promoter is correct. Obtaining an improved pBA002 vector with replaced promoters: promoter ole-pBA 002.

Transforming agrobacterium GV3101 competence by extracting plasmid and electric shock, sequencing to obtain positive strain, and storing at-80 deg.c.

2. Transformation of Arabidopsis thaliana by flower dipping method

(1) Agrobacterium infection of Arabidopsis thaliana

a. Arrangement of conversion media

Transformation medium composition: 1/2MS culture medium, 0.01. mu.g/mL BAP, 5% sucrose, 0.02% silwet L-77, adjusted to pH 5.7 with KOH.

b. Activated monoclonal positive Agrobacterium GV3101 strain containing the desired gene plasmid was picked to 5mL fresh YEP liquid medium containing Kan (50mg/L) and rifampicin (50mg/L) at 28 ℃ for 24h at 210 g/min.

c. 1mL of the bacterial liquid is taken to be inoculated into 100mL of fresh YEP liquid culture medium containing Kan (50mg/L) and rifampicin (50mg/L), the temperature is 28 ℃, 210g/min and 7h are carried out, and the OD value is about 0.8.

d. And (3) taking 50mL of the bacterial liquid, placing the bacterial liquid in an EP tube, centrifuging for 5min at room temperature at 5000g/min, removing supernatant, and carrying out heavy suspension precipitation by using a transformation medium until the OD value reaches about 0.8.

e. Arabidopsis plants to be transformed were laid flat and the bud part was inserted into a 50mL EP tube containing transformation medium and dip-stained for 10 s. The invaded liquor was gently blotted with absorbent paper. After dark culture for 16-24h, normal culture is resumed.

(2) Screening of Positive plants

And (3) the harvested transgenic plant seeds are planted on an MS culture medium containing 25mg/L Kanamycin, and the transformed seedlings with resistance which grow for about ten days are selected and transplanted into soil to continue growing. Extracting genome DNA of wild plants and transgenic plants, identifying whether the transgenic plants are transgenic positive plants by PCR, respectively harvesting seeds of each plant, further planting the seeds on an MS culture medium containing 50 mu g/mL of Kanamycin, selecting Kanamycin resistance to screen resistant transformation seedlings with a separation ratio of 3:1, transplanting the resistant transformation seedlings into soil to grow, respectively harvesting seeds of each plant, further planting the seeds on the MS culture medium containing 50 mu g/mL of Kanamycin, and obtaining the transformation seedlings with 100 percent of Kanamycin, namely transgenic positive homozygote plants.

(3) Detection of GUS Gene tissue expression Activity

And (3) carrying out GUS tissue staining on arabidopsis positive homozygote transformed by the constructed seed specific promoter expression vector at each development stage, selecting 10-day seedlings, 16-day seedlings, 25-day seedlings, young stem leaves and flowers, pods, seeds and pods 5 days after flowering, 8 days after flowering, 11 days after flowering, 14 days after flowering and 20 days after flowering, putting GUS staining solution into the seeds and the pods, and staining for different times according to different tissues and organs, wherein the staining time is also different, and the staining time for general seeds and pods is longer. After all tissues are decolorized by ethanol after dyeing, the tissues are transparent by transparent liquid (the tissues of seedlings, root tips and the like are transparent for about 5-l0 min; the tissues of pods, seeds and the like need to be transparent for several hours or even overnight), and then the tissues are observed by a stereoscopic microscope and photographed.

The dyeing liquid formula comprises: 1) X-Gluc mother liquor: X-Gluc, 20mM stock solution prepared with N-N-Dimethylformamide (DMF), was dispensed and stored at-20 ℃. 2) X-Gluc base fluid (50mM PBS, pH 7.0): 50mM NaH₂PO₄，50mM Na₂HPO₄Dissolving together; 10mM Na₂EDTA，0.1％Triton-100，1mM K₃[Fe(CN)₆]，0.5mM K₄[Fe(CN)₆]。

FIG. 1.B shows the result of GUS staining, which indicates that the GUS gene is expressed only in immature seeds and the germination stage of the seeds, but not substantially in other tissues, and indicates that the obtained promoter of the oil body protein gene is a seed-specific promoter.

Example 2 acquisition of GmDGAT2A seed-specific overexpression transgenic Soybean

Firstly, obtaining a recombinant plasmid promoter ole GmDGAT2A-pBA002

1. Primers were designed to amplify the promoter sequence of interest and oil body protein promoter amplification was performed using the soybean total DNA described in example 1. NcoI and XbaI enzyme cutting sites are added, and amplification primers are as follows:

Forward：5’-CCATGGTTCTATTCAGGAGGTGGTTG-3’(SEQ ID NO：8)；

Reverse：5’-TCTAGAGAGTTTTGAGTGAAGAGTGAG-3’(SEQ ID NO：9)；

the amplified sequence size is 1601 bp. After purification of the PCR product, the PCR product and pBA002 plasmid were digested with NcoI and XbaI enzymes at 37 ℃ for 3 hours, and the desired fragment was recovered. The digested PCR product fragment was ligated into the plasmid cleavage site using T4 ligase (purchased from TAKARA) at 4 ℃ overnight. The ligation products were transformed into E.coli competent cells, recovered at 37 ℃ for 1h and plated overnight for culture. And (3) selecting a single colony quality-improving particle for sequencing, verifying that the sequence of the target promoter is correct, and storing the target plasmid promoter ole-pBA 002.

2. Primers were designed to amplify the target gene GmDGAT2A, and soybean GmDGAT2A target gene amplification was performed using the soybean cDNA described in example 1. Xba I and SpeI restriction sites were added, and the amplification primers were:

Forward：5’-TCTAGAATGCAGCGCACGGCGGCGGCGAC-3’(SEQ ID NO：10)；

Reverse：5’-ACTAGTTCAAACAATTCTCAACTCAAGGTTTG-3’(SEQ ID NO：11)；

the amplified sequence size was 1014 bp. The PCR product and the aforementioned promoter ole-pBA002 plasmid were simultaneously digested with Xba I and SpeI enzymes. And (3) utilizing T4 ligase to ligate the digestion products, transforming the ligation products into escherichia coli competent cells, recovering for 1h at 37 ℃, and plating for overnight culture. And (3) selecting a single colony quality-improving particle for sequencing, verifying that the sequence of a target promoter is correct, and storing a target plasmid promoter ole, namely GmDGAT2A-pBA 002. The recombinant vector is shown in FIG. 2.

3. Transforming agrobacterium GV3101 competence by electric shock of extracted plasmid promoter ole GmDGAT2A-pBA002, sequencing to obtain positive strain, and storing at-80 deg.C for use.

II, obtaining of GmDGAT2A seed specific overexpression transgenic soybean

1. Method for transforming soybean by using cotyledonary node infection method

a) Media used for soybean transformation:

GM culture medium: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.59g/L of MES, 3.5g/L of Phytagel, and autoclaving for 20 minutes at 5.8,121 ℃ by adjusting the pH value with KOH;

IF medium: 0.321g/L of B5 culture medium, 30g/L of sucrose, 3.9g/L of MES, adjusting the pH value to 5.4,121 ℃ by KOH, carrying out autoclaving for 20 minutes, and adding 30.25mg/L of GA30, 1.67mg/L of 6-BA and 40mg/L of AS;

CCM medium: 0.321g/L of B5 culture medium, 30g/L of sucrose, 3.9g/L of MES, 10g/L of agarose, adjusting the pH value to 5.4,121 ℃ by KOH, autoclaving for 20 minutes, and adding 30.59mg/L of GA30, 1.67mg/L of 6-BA, 40mg/L of AS, 154.2mg/L of DTT, 400mg/L of L-Cys and 158mg/L of NaThio;

SIW medium: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.59g/L of MES, 1.67mg/L of 6-BA, 100mg/L of Cef and 500mg/L of Carb after the pH value is adjusted to 5.8,121 ℃ and the autoclave is sterilized for 20 minutes;

SEM culture medium: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.59g/L of MES, 3.5g/L of Phytagel, 1.67mg/L of 6-BA, 100mg/L of Cef and 500mg/L of Carb after the pH value is adjusted to 5.8,121 ℃ and the mixture is autoclaved for 20 minutes by KOH;

SIM culture medium:

1) a large screening stage: 3.21g/L of B5 culture medium, 30g/L of sucrose, 0.59g/L of MES, 3.5g/L of Phytagel, 1.67mg/L of 6-BA, 100mg/L of Cef, 500mg/L of Carb and 6mg/L of Glufo after the pH value is adjusted to 5.8,121 ℃ and the mixture is autoclaved for 20 minutes;

2) and (3) secondary screening stage: MSB4.44g/L, sucrose 30g/L, MES0.59g/L, agar powder 10g/L, KOH to adjust pH to 5.8,121 deg.C, autoclaving for 20 minutes, adding ZR 1mg/L, IAA 0.1mg/L, Asn 50mg/L, L-Pyro 100mg/L, GA₃0.5mg/L,Cef 100mg/L,Carb 500mg/L,Glufo 3mg/L；

3) A subculture stage: MSB4.44g/L, sucrose 30g/L, MES0.59g/L, agar powder 10g/L, KOH regulation to 5.8,121 ℃ and autoclaving for 20 minutes, ZR 1mg/L, IAA 0.1mg/L, Asn 50mg/L, L-Pyro 100mg/L, GA30.5mg/L, Cef 100mg/L, Carb 500mg/L and Glufo 1.5mg/L are added;

RM medium: 2.22g/L of MSB culture medium, 30g/L of sucrose, 0.59g/L of MES, 10g/L of agar powder, adjusting the pH value to 5.8,121 ℃ by KOH, and carrying out autoclaving for 20 minutes;

b) soybean transformation

(1) Obtaining of sterile seedlings

Selecting soybean seeds with smooth surfaces, no cracks and different colors and uniform and full sizes, putting the seeds into a 50mL centrifuge tube, adding a proper amount of 0.1% mercuric chloride disinfectant, inverting the centrifuge tube for several times to fully contact the seeds with the disinfectant, cleaning for 7-8 minutes, and pouring out the disinfectant. Then, adding proper amount of sterilized water to clean for three times, each time for 7-8 minutes. Inoculating the sterilized seeds on a GM culture medium, and culturing at 25 ℃ under illumination for 16h every day.

(2) Preparation of cotyledonary node explants

Taking a sterile soybean seedling which grows for 5-6 days, removing seed coats, cutting off hypocotyls, and leaving 3-5 mm; longitudinally cutting and separating two cotyledons along hypocotyls, scraping terminal buds and primary buds, cutting off the expanded cotyledonary nodes by using a surgical knife for ten times, wherein the knife edge depth is about 0.5mm, the knife edge cannot be too deep, otherwise, a growing point can be injured, and the knife edge cannot be too shallow, otherwise, agrobacterium cannot be infected into the growing point.

(3) Infection and co-culture of explants

Selecting Agrobacterium to culture in YEP culture medium at 28 deg.C and 200rpm overnight to obtain OD₆₀₀Centrifuging the bacterial solution with a value of about 1.0-1.2 at 4000rpm for 10min to collect thallus, and suspending the thallus with IF culture medium to obtain OD₆₀₀The re-suspended bacterial liquid with the value of about 0.5-0.6 is used as the infection liquid. Thereafter, the prepared cotyledonary node explants were placed in an infection solution and infected at 28 ℃ for 45min at 150 rpm. The invaded solution was poured off, and the explants were spread on CCM medium with a layer of sterile filter paper with the paraxial side down, and co-cultured in the dark at 25 ℃ for 5 days.

(4) Bud induction culture

After 5 days of co-culture, the explants were transferred to a sterile culture tank, washed with SIW medium with appropriate amounts of 100mg/L Cef and 500mg/L Carb at 25 deg.C for 45min at 150rpm, after the wash solution was decanted, washed twice with clean SIW medium in a clean bench until the wash solution was clear, and the explants were placed in a large petri dish with sterile filter paper, and excess liquid was aspirated.

After the seeds were dried, cotyledons were placed paraxially downward, and the hypocotyls were inserted obliquely into the SEM medium and cultured at 25 ℃ under illumination for 16h a day for two weeks.

(5) Stage of shoot screening

A large screening stage: culturing the cotyledonary node in SEM culture medium for two weeks, selecting cotyledonary node with adventitious bud, transferring to SIM culture medium containing 6mg/L screening agent-glufosinate-ammonium, culturing at 25 deg.C under 16h per day for two weeks;

and (3) secondary screening stage: after two weeks, the explants in the large-sieve culture medium are transferred to an SIM culture medium containing 3mg/L phosphinothricin, and the necrotic tissues at the stem of the hypocotyl are scraped off by a scalpel before the explants are transferred to ensure that the tissues absorb the nutrients of the culture medium, and the explants are cultured for two weeks under the illumination of 16h every day at the temperature of 25 ℃;

a subculture stage: after large screening, the surviving cotyledonary nodes were transferred to SIM medium containing 1.5mg/L phosphinothricin, and transferred to new medium every two weeks, while the aged tissue of hypocotyl stem was still cut off during transfer.

(6) Rooting and transplanting

After several subcultures, when the height of the regenerated seedling is 2-3cm, transferring the regenerated seedling from a culture dish into a culture tank, after the seedling grows to be more than 5cm, cutting the extended resistant bud extension base, transferring the cut resistant bud extension base into an RM culture medium, and inducing rooting under the condition of illumination for 16h every day at 24 ℃.

After two weeks, the rooted whole plantlets were acclimatized for 3 days, cleaned of the root medium, transplanted to vermiculite: culturing in a flowerpot with nutrient soil of 1:5 in an incubator at 25 ℃ for 16h under illumination until the plant is mature and pod-bearing.

2. Material identification and generation

a) Identification of transgenic Soybean at DNA level

The target gene of the soybean transgenic plant PCR detection is a bar fragment of 459bp coding screening agent resistant protein on a carrier, and a primer is designed according to the bar gene fragment:

Forward：5′-GTACCGGCAGGCTGAAGTCC-3′(SEQ ID NO：12)；

Reverse：5′-CGGTCTGCACCATCGTCAAC-3′(SEQ ID NO：13)。

PCR was performed using soybean genomic DNA extracted in the previous stage as a template and wild type P03 as a control. The enzyme used for amplification is TaqDNA polymerase.

And selecting positive seedlings for propagation and generation planting. And carrying out PCR identification on resistance genes of the offspring seedlings until no resistance separation exists, thus obtaining the homozygous transgenic material. As shown in fig. 3A.

b) Identification of transgenic Soybean at RNA level

Young seeds (30DAF) and leaves of wild type soybean (P03) and homozygous transgenic soybean material were collected and subjected to RNA extraction and reverse transcription experiments as described in example 1, and RT-PCR experiments were performed using the obtained cDNA to detect changes in the expression level of GmDGAT2A in the leaves and seeds of transgenic material; the primers used were:

Forward：5′-TTGTGGCTCGGAACCATTCA-3′(SEQ ID NO：14)；

Reverse：5′-TGAGCACGAAAAGCAAACCG-3′(SEQ ID NO：15)。

as shown in fig. 3B, comparative analysis of the expression amount of GmDGAT2A in leaves and seeds of wild-type soybean and transgenic soybean revealed: the expression of GmDGAT2A in soybean seeds is promoted by using an oil body protein promoter, the expression quantity of GmDGAT2A in the seeds is specifically improved, and the expression quantity of GmDGAT2A in leaves is not improved.

Example 3 statistical analysis of seed oil content of GmDGAT2A seed-specific over-expressed transgenic Soybean

Statistical analysis of oil content of GmDGAT2A seed specific overexpression transgenic soybean seeds

1. Oil extraction from mature seeds

(1) Respectively picking plump soybean seeds with similar sizes of P03(WT) and over-expression (OE1 and OE2), and crushing by using a plant sample crusher; the ground sample with the fineness of 0.3mm (50 meshes) to 1mm is screened by using a national standard sieve to obtain about 15mg of the ground sample, the weight of the ground sample is recorded, and the ground sample is placed in a 15mL pointed-bottom centrifuge tube for fat extraction.

(2) Adding 2mL of isopropanol containing 0.01% BHT into each tube of sample weighed in the step (1), and carrying out water bath at 85 ℃ for about 30 min; 3mL of n-hexane were added and the number was reversedMixing completely, and standing at room temperature for 5 min; 2.5mL of 15% Na was added to the mixture₂SO₄Turning over for several times to mix completely, standing at room temperature for 5min, and separating the mixture into three layers; sucking the uppermost layer into a 20mL glass bottle; adding 4mL of n-hexane/isopropanol 7:2(v/v) into the rest mixed solution, reversing the mixture for several times until the mixture is completely mixed, and standing the mixture at room temperature for 10 min; collecting the uppermost layer to the previous glass bottle; drying the grease extracting solution in the glass bottle by using nitrogen, and keeping the grease at the bottom of the centrifugal tube to be yellow; 2mL of methanol (containing 5% H) was added to the dried oil₂SO₄And 0.01% BHT), adding 30 μ L of 5.4mM 17:0TAG as internal standard, heating in water bath at 95 deg.C for 2 h; and (3) after water bath, standing and cooling, adding 1mL of water and 1mL of n-hexane into a glass bottle, reversing for several times to completely mix the mixture, standing for 5min, centrifuging at the room temperature of 4500rpm for 5min, taking the upper organic phase, transferring the upper organic phase into a gas phase measuring bottle, and using the upper organic phase for gas phase measurement.

2. Methyl esterification and gas chromatography detection analysis

The samples and standards were measured by Gas Chromatography (GC). The type of chromatographic column used was SpTM-2560,100 m.times.0.25 mm,0.2um FILM, the injection volume was 1. mu.L, and the temperature at the column port was 260 ℃.

The results of the oil and fat determination of the soybean seeds can be seen: overexpression of GmDGAT2A in soybean seeds significantly increased the oil content (see FIG. 4).

As can be seen from the analysis of the oil components, the over-expression of GmDGAT2A in the soybean seeds obviously improves the linoleic acid (C18:2) content in the oil (as shown in figure 5)

Second, statistics of agronomic traits of transgenic soybean field specifically overexpressed by GmDGAT2A seeds

The above-mentioned soybean materials T4 and T5 were planted in test fields of the jilin academy of agricultural sciences and subjected to statistics of relevant agronomic traits, as shown in table 1. From the statistical results it can be seen that: the GmDGAT2A is over-expressed in soybean seeds, the agronomic characters of plant height, branch number, knot number, single plant pod number and the like are not changed, and the growth of the plant is not influenced.

Table 1 shows the statistical results of the agronomic characters of the transgenic soybean material in the field

Line of plants

Oil content (%)

Protein (%)

Plant height (cm)

Number of knots

Number of branches

Number of pods per plant

WT

20.15±0.343

41.55±0.49

85.4±8.15

18.8±1.64

1.3±0.55

94±13.55

OE-1(2018)

22±0.71

40.55±0.78

86.4±6.02

18.4±1.95

0.8±1.79

87.4±38.08

OE-1(2019)

22.25±0.77

40.05±3.60

83.2±4.87

17±1.87

1.8±0.45

94.2±25.40

OE-2(2018)

21.7±0.42

40±0.42

82±3.81

17.6±0.89

1.8±2.49

104.6±39.78

OE-2(2019)

21.07±0.11

43.4±0.36

86.8±5.93

18.8±1.10

1.4±0.55

96±22.81

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.

Sequence listing

<110> Nanjing university of agriculture

Application of <120> GmDGAT2A in increasing content of soybean oil and content of linoleic acid

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1601

<212> DNA

<213> Soybean (Glycine max)

<400> 1

ttctattcag gaggtggttg ggttttgcta tggtgcagca caatgaagtt cagcagcatt 60

atttgcaaca tggtcttctg tttcggggta agaaatttcg taggtttcct tatttgtttt 120

ggcacgctct atgaagcaca attacatctt tcttgttcaa agtgtcttta tgtccgacat 180

atttctaata ccagcatatt caatactgtt cgatatttat ctgacatttt attgaatttt 240

ttttttacgt aaacactcaa actatcacct ttacataatt aatacactta aaaaatataa 300

aatattaatt ttaaaaaata aaatatatca tcaatttaca tattttgttc tatattattc 360

atgttttatt taattaaaat ctttttgttt gttagtgtct tatattttaa atattaacca 420

tattgaaata tttgtgtggt gttcgatatc ggtatcatag tcggtgcttc acagggcatg 480

ctatttgcga gtgtctatgg actatggttg ctgcggaata aagtggtttt ccaaaatgtg 540

atttcggatg tgtatggtat gctgtcgcac atccgacgta tttcttggat ttggctatac 600

agggtcccct tctctggtag tggtggtgta gtgtttggaa actggtgtat ttgtcccctg 660

gattgcttac ggtgtgtatg ttagagaccc tattatgctc ctttattacg atctgatgta 720

atctttattg tatttgggtc acattttatg gctcttaata atagtatttg attttaattt 780

tcagaaagaa aaaaaaacaa aatagtgtgt ataaaattat tataaacttt tagtatatat 840

atatatatat atgattctta cttataaaaa aataattaca ccaataaatt catcctcaaa 900

tattacgtta tgaaatcaga gctattttag ttatgcatat gcaaatgtct taattttttt 960

ttcttaacct atttttttta tttgctcttc tatataaaat cactctaata agattgtctt 1020

cgctggagtt tacctgtaac ttataccaaa aattataaaa tcgctctaag ggaagatatg 1080

agtatgactg atcccttgtt atattcatgc aaattatatg gtgtgttcgt tctgatataa 1140

atcgataacg tttagtggat ataattgtta gagaaagtag aagccttatc ttatcttggt 1200

atgttaaaac ggttttatta cattttctat cattgcaatt aatcattaaa caaaaacaga 1260

aaatcctagc acataacata tatatgaaca taaccataga agagcggcac gtacatatgt 1320

tggcctagga tcgttgttaa gtgttaacgc tggtccaaaa catgcaacaa acaacaacca 1380

agaaaaaaaa aaaaaaggta cgtacaaaaa acctaacgtg tcatcaaaca catgcatggg 1440

ttttgcatgc aagccttgca tgaaaagctt gccaacacgt gccaaaccac ctcctcaggt 1500

gttgccaccc aagcctccac tcaccaattt ctccatttat accctcatta ccaccacctt 1560

aaaccctacc acattaatta ctcactcttc actcaaaact c 1601

<210> 2

<211> 1014

<212> DNA

<213> Soybean (Glycine max)

<400> 2

atgcagcgca cggcggcggc gacagatgaa ccacggcggc gctccggcga cgcggagggg 60

gagaaggtgt tcaaggggag cgaggtgttc ggcgacacgt caccaaatta cttgaagacc 120

attttggccc tggcgttgtg gctcggaacc attcatttca acgtcgcgtt ggtgctcttc 180

gcaatcttct tcctctctct ccacaaagca cttttgcttt tcggtttgct tttcgtgctc 240

atggtaattc ctgttgatga aaagagcaaa ttcggtcgaa aattatccag gtacatatgc 300

aagcacgttt gcgcttactt tcccatcacg cttcatgtgg aggacatgaa ggcctttcat 360

cccaatcgtg cttatgtttt tggttatgaa ccacattcag ttttgccaat tggagttgtt 420

gcgttagctg acaacacagg ttttatgcct cttcctaaaa taaaagttct tgctagcagt 480

gcgatatttt acacaccatt tttgagacac atatggacat ggttgggctt aacaccagtg 540

acaaggaaaa ggtttacctc cctgttggat gctggctata gttgtatctt gatacctggt 600

ggagtgcaag aagcatttct catggagcat ggttctgaga ttgcctatct taaagcaaga 660

aggggatttg tccgcatagc aatggagaaa ggaaaacccc tggttccagt tttctgcttt 720

ggtcagtcaa atgtctataa gtggtggaaa ccaggtggga agttaattct gaattttgca 780

agggctgtca agttctcccc aatatatttt tggggaattt ttggatctcc gatacccttt 840

aaacatccga tgcatgtggt ggtgggtaga ccaattgagc tcgagaaaaa tcacgagcca 900

actcctgagg aggttgccag aatacatagc caatttgttg aagcacttca agatctattt 960

gaacgacaca aagctcgagc tggatatcca aaccttgagt tgagaattgt ttga 1014

<210> 3

<211> 337

<212> PRT

<213> Soybean (Glycine max)

<400> 3

Met Gln Arg Thr Ala Ala Ala Thr Asp Glu Pro Arg Arg Arg Ser Gly

1 5 10 15

Asp Ala Glu Gly Glu Lys Val Phe Lys Gly Ser Glu Val Phe Gly Asp

20 25 30

Thr Ser Pro Asn Tyr Leu Lys Thr Ile Leu Ala Leu Ala Leu Trp Leu

35 40 45

Gly Thr Ile His Phe Asn Val Ala Leu Val Leu Phe Ala Ile Phe Phe

50 55 60

Leu Ser Leu His Lys Ala Leu Leu Leu Phe Gly Leu Leu Phe Val Leu

65 70 75 80

Met Val Ile Pro Val Asp Glu Lys Ser Lys Phe Gly Arg Lys Leu Ser

85 90 95

Arg Tyr Ile Cys Lys His Val Cys Ala Tyr Phe Pro Ile Thr Leu His

100 105 110

Val Glu Asp Met Lys Ala Phe His Pro Asn Arg Ala Tyr Val Phe Gly

115 120 125

Tyr Glu Pro His Ser Val Leu Pro Ile Gly Val Val Ala Leu Ala Asp

130 135 140

Asn Thr Gly Phe Met Pro Leu Pro Lys Ile Lys Val Leu Ala Ser Ser

145 150 155 160

Ala Ile Phe Tyr Thr Pro Phe Leu Arg His Ile Trp Thr Trp Leu Gly

165 170 175

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

180 185 190

Tyr Ser Cys Ile Leu Ile Pro Gly Gly Val Gln Glu Ala Phe Leu Met

195 200 205

Glu His Gly Ser Glu Ile Ala Tyr Leu Lys Ala Arg Arg Gly Phe Val

210 215 220

Arg Ile Ala Met Glu Lys Gly Lys Pro Leu Val Pro Val Phe Cys Phe

225 230 235 240

Gly Gln Ser Asn Val Tyr Lys Trp Trp Lys Pro Gly Gly Lys Leu Ile

245 250 255

Leu Asn Phe Ala Arg Ala Val Lys Phe Ser Pro Ile Tyr Phe Trp Gly

260 265 270

Ile Phe Gly Ser Pro Ile Pro Phe Lys His Pro Met His Val Val Val

275 280 285

Gly Arg Pro Ile Glu Leu Glu Lys Asn His Glu Pro Thr Pro Glu Glu

290 295 300

Val Ala Arg Ile His Ser Gln Phe Val Glu Ala Leu Gln Asp Leu Phe

305 310 315 320

Glu Arg His Lys Ala Arg Ala Gly Tyr Pro Asn Leu Glu Leu Arg Ile

325 330 335

Val

Claims (10)

1. The application of the GmDGAT2A protein is selected from S1) or S2):

   s1) regulating fatty acid synthesis in plant tissues;

   s2) cultivating transgenic plants with high oil and high linoleic acid;

   the GmDGAT2A protein is selected from a1) or a2) or a 3):

   a1) the amino acid sequence is SEQ ID NO: 3;

   a2) in SEQ ID NO: 3, the N end or/and the C end of the protein shown in the formula 3 is connected with a label to obtain a fusion protein;

   a3) converting SEQ ID NO: 3 through substitution and/or purpose or deletion and/or addition of one or more amino acid residues, and/or the protein is obtained and is related to the regulation and control of fatty acid synthesis in plant tissues.
2. 2. Use of a nucleic acid molecule encoding the GmDGAT2A protein of claim 1, selected from S1) or S2):

   s1) regulating fatty acid synthesis in plant tissues;

   s2) cultivating transgenic plants with high oil and high linoleic acid.
3. 3. The use according to claim 2, wherein the nucleic acid molecule is a DNA molecule as represented by b1) or b 2):

   b1) the coding region is SEQ ID NO: 2;

   b2) a DNA molecule which is hybridized with the nucleotide sequence limited by b1) under strict conditions and codes the GmDGAT2A protein.
4. 4. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising a nucleic acid molecule encoding the GmDGAT2A protein of claim 1, selected from S1) or S2):

   s1) regulating fatty acid synthesis in plant tissues;

   s2) cultivating transgenic plants with high oil and high linoleic acid;

   the nucleic acid molecule is a DNA molecule shown as b1) or b 2):

   b1) the coding region is SEQ ID NO: 2;

   b2) a DNA molecule which is hybridized with the nucleotide sequence limited by b1) under strict conditions and codes the GmDGAT2A protein.
5. 5. The use of any one of claims 1 to 4, wherein said modulating fatty acid synthesis in plant tissue increases oil content and linoleic acid content in the plant or decreases oil content and linoleic acid content in the plant; preferably, the tissue is a seed.
6. 6. The use according to any one of claims 1 to 5, wherein the plant is c1) or c 2): c1) a dicotyledonous plant; c2) a monocot plant; preferably, the dicotyledonous plant is a crucifer or a leguminous plant; more preferably, the crucifer is arabidopsis thaliana, preferably a wild type arabidopsis thaliana Columbia-0 subtype; more preferably, the leguminous plant is soybean.
7. 7. A method for cultivating a transgenic plant with high oil content and high linoleic acid content, comprising the following steps: improving the expression quantity and/or activity of the GmDGAT2A protein in the starting plant to obtain a transgenic plant; compared with the original plant, the neutron oil content and the linoleic acid content of the transgenic plant are improved.
8. 8. The method as claimed in claim 7, wherein the effect of increasing the expression level and/or activity of GmDGAT2A protein in the starting plant is achieved by changing a promoter.
9. 9. The method as claimed in claim 8, wherein the promoter is a seed specific promoter ole1, which is used for promoting GmDGAT2A to express specifically in the seeds of transgenic materials, and improving the expression amount and activity of GmDGAT2A under the dead weight; preferably, the nucleotide sequence of the promoter is as shown in SEQ ID NO: 1 is shown.
10. 10. A plant breeding method, which increases the content of plant oil and the content of linoleic acid components by increasing the expression level of GmDGAT2A in the plant as claimed in claim 1.

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