Application of coix lacryma-jobi diacylglycerol acyltransferase gene ClDGAT2

Abstract

The invention relates to the field of biology, in particular to application of a coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2). The invention discloses an application of a coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2) in improving the yield of oil. The coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2) has the following functions: triglyceride can be generated, a DGAT enzyme function is realized, and the capability of recovering TAG defect type saccharomyces cerevisiae oil production is realized.

Description

Technical Field

[0001] The present invention relates to the field of biology, and specifically relates to the application of the Coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2). Background Art

[0002] Coix lacryma-jobi, belonging to the genus Coix of the Gramineae family, is a plant with a long history of being both a medicine and a food. The fruit of Coix lacryma-jobi L. is often used as a good medicine for dispelling dampness, relieving arthralgia, strengthening the spleen and stopping diarrhea, and expelling pus and detoxifying. In recent years, the research and clinical treatment of the active ingredient in Coix lacryma-jobi, Coix seed oil, have proved that Coix seed oil has very significant anti-cancer effects and the effect of enhancing the efficacy of chemotherapy.

[0003] Coix seed oil extracted from Coix lacryma-jobi has valuable anti-cancer value. Therefore, in order to popularize the functions of Coix seed oil, expand its usage, and increase its oil yield, it is very crucial.

[0004] With the development of technology, more and more research has started to find breakthroughs from the level of microbial synthetic biology, using microorganisms to synthesize compounds with low yields in reality or studying the compound synthesis process in microorganisms.

[0005] Triglyceride (TAG) is composed of three molecules of fatty acid and one molecule of glycerol, and is widely present in organisms. The main storage form of oil in most plant seeds is also triglyceride, which is generally catalyzed and synthesized by isozymes encoded by similar genes, and is also an important energy source required during the germination of plant seeds and the development of seedlings. Due to different substrates, the TAG biosynthesis pathway is divided into the Kennedy pathway and the acyl-CoA-independent pathway (PDAT pathway). In the process of plant TAG synthesis, both of these pathways play important roles, and their key rate-limiting enzymes are DGAT and PDAT.

[0006] The main function of diacylglycerol acyltransferase (DGAT) is to catalyze the synthesis of triglyceride, and it is the only rate-limiting enzyme in the Kennedy pathway.

[0007] H1246 yeast is obtained by knocking out four genes in the yeast genome through genetic engineering means: the DGA1 gene encoding diacylglycerol acyltransferase, the LRO1 gene encoding phospholipid diacylglycerol acyltransferase, and the ARE1 and ARE2 genes encoding different sterol acyltransferases. In recent years, H1246 yeast has been widely used as an engineering bacterium for studying the TAG synthesis pathway. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide an application of Coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2).

[0009] To solve the above problems, the present invention provides an application of Coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2) in increasing the oil yield.

[0010] As an improvement of the application of the present invention: it can produce triglyceride.

[0011] As a further improvement of the application of the present invention: it has the function of DGAT enzyme.

[0012] As a further improvement of the application of the present invention: it has the ability to restore oil production in TAG-deficient Saccharomyces cerevisiae.

[0013] As a further improvement of the application of the present invention: the Coix lacryma-jobi diacylglycerol acyltransferase gene (ClDGAT2) is respectively the following 10 genes:

[0014] ClDGAT1-1, ClDGAT1-2 (preferred), ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3 (preferred), ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, ClWS / DGAT2-4;

[0015] The corresponding sequences are shown as no1 to no10 in the sequence listing.

[0016] As a further improvement of the application of the present invention:

[0017] 1), Each Coix diacylglycerol acyltransferase gene (ClDGAT2) was respectively operated as follows: ligated to the pYes2 vector and transformed into DH5α Escherichia coli competent cells. After PCR, the positive bacteria with the correct band positions were amplified and cultured, and plasmids were extracted to obtain the corresponding ClDGAT2 gene recombinant plasmids (each ClDGAT2 obtained one ClDGAT2 gene recombinant plasmid);

[0018] 2), The ClDGAT gene recombinant plasmid was transformed into H1246 (TAG-deficient Saccharomyces cerevisiae mutant strain H1246); cultured with SC-Ura solid inhibitory medium, and after screening, the obtained positive yeast cells containing the ClDGAT gene recombinant plasmid were amplified and cultured to obtain a bacterial solution for oil extraction.

[0019] That is, the positive yeast cells all restored the ability to synthesize TAG, indicating that the proteins encoded by these 10 DGAT genes have the function of DGAT enzyme.

[0020] As a further improvement of the application of the present invention:

[0021] Yeast strains transfected with the ClDGAT2-2, ClDGAT3 and ClWS / DGAT2-4 gene fragments produced high levels of fatty acids;

[0022] Yeast strains transfected with the ClDGAT1-2, ClDGAT2-2 and ClDGAT3 gene fragments produced high levels of fat.

[0023] The specific scheme of the present invention is as follows:

[0024] 1. By searching the CDS sequences of DGAT genes of Arabidopsis thaliana, Zea mays and Sorghum bicolor in NCBI and performing homologous comparison with the Coix genome, 10 Coix DGAT gene family genes were screened and named in this article as: ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-2, ClDGAT2-1, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, ClWS / DGAT2-4.

[0025] Total RNA was extracted from Coix lacryma-jobi using molecular biology techniques, reverse transcribed into cDNA, and primers were designed to amplify a total of 10 DGAT genes of the Coix DGAT gene family from it. That is, these 10 gene fragments were obtained by reverse transcribing Coix total RNA into cDNA. The results showed that these 10 genes were all expressed in Coix lacryma-jobi RNA.

[0026] 2. The 10 obtained DGAT genes were ligated to the pYes2 plasmid and transformed into competent DH5α Escherichia coli cells, which were then spread on 10 LB plates with Amp resistance and cultured overnight. The next day, single colonies of Escherichia coli cells were picked and identified by PCR for positivity. The positive bacteria with the correct band positions were cultured on a large scale and then plasmids were extracted. The results showed that the competent Escherichia coli cells had well received the transformed plasmids, and the designed primers also had high specificity. Finally, the extracted plasmids also had high stability and could be used for the transformation of competent yeast cells.

[0027] 3. Ten kinds of plasmids were used to transform competent yeast cells. After culturing with SC-Ura solid inhibitory medium, single colonies of yeast cells were picked and identified for positivity, and the positive yeast cells were cultured on a large scale. As a result, 10 kinds of positive yeast cells and the bacterial liquid that could be used for oil extraction after large-scale culture were obtained.

[0028] 4. For each of the enlarged culture broths of the 10 obtained positive yeast cells, 1 ml of the bacterial liquid was left for Nile red staining, and the rest was used to prepare dry yeast powder. 10 portions of 50 mg were respectively weighed for fatty acid extraction; 10 portions of 20 mg were taken for fat extraction and TCL chromatography. The results showed that through Nile red staining, it was proved that all 10 positive yeasts produced oil. The results of TCL chromatography showed that each yeast produced triglyceride, and the trace of ClDGAT3 was particularly obvious, indicating that the proteins encoded by the 10 genes had the function of DGAT enzyme and restored the ability of yeast to synthesize TAG. The fat yield and fatty acid yield of the yeast bacterial liquid containing the ClDGAT3 gene were both relatively high, indicating that it played a major oil-producing role in the oil production of Coix lacryma-jobi.

[0029] In the present invention:

[0030] The present invention uses seamless cloning technology to construct 10 kinds of recombinant vectors, which are transformed into the TAG-deficient Saccharomyces cerevisiae mutant strain H1246 for yeast functional complementation testing and yeast oil extraction.

[0031] The results showed that: (1) The primers used to amplify 10 DGAT genes had good specificity, and all 10 DGAT genes were expressed in Coix lacryma-jobi. (2) After the pYes2 plasmids ligated with 10 DGAT gene fragments were used to transform H1246 yeast competent cells respectively, the observation results of thin-layer chromatography and Nile red staining showed that all 10 positive yeasts restored the ability to synthesize TAG, indicating that the proteins encoded by these 10 DGAT genes had the function of DGAT enzyme. (3) The results of fatty acid extraction and fat extraction in 10 positive yeasts showed that the ClDGAT3 gene had a high fatty acid and fat extraction rate, indicating that it played a major role in the synthesis of Coix lacryma-jobi oil components, which had guiding significance for improving the yield of Coix lacryma-jobi oil and Coix lacryma-jobi breeding (the experiments of the present invention characterized the functions of the Coix lacryma-jobi DGAT gene family in vitro and laid a foundation for Coix lacryma-jobi breeding).

[0032] In the present invention, the oil production rates of the DGAT gene family encoding key enzymes in the TAG pathway of the medicinal plant Coix lacryma-jobi were mainly measured and compared. From the results, all DGAT genes had the ability to restore oil production in yeast, but there were differences in oil production efficiency: among them, the oil production efficiencies of ClDGAT3 and ClDGAT1-2 were relatively high, while the oil production efficiencies of ClDGAT1-1 and ClDGAT2-1 were relatively low. The efficiencies of the DGAT enzymes encoded by different DGAT genes in synthesizing oils were not the same.

[0033] In the present invention, oils were successfully extracted from all the positive H1246 yeasts transformed with ten pYes2 plasmids containing different DGAT gene fragments; after the positive yeast cells were stained with Nile red, oils were stained, and TCL also proved the production of triglycerides and diglycerides, indicating that the expression of these 10 genes successfully restored the ability of yeast to synthesize TAG. Among them, ClDGAT3 had a relatively high content ratio in both fatty acid extraction and fat extraction, indicating that the enzyme encoded by it played a major role in the TAG synthesis pathway of Coix lacryma-jobi. The yields of yeast oils and fatty acids into which the ClDGAT2-1 gene was transferred were relatively low, indicating that ClDGAT2-1 had a weak ability to synthesize oils. In addition, the fatty acid content was consistent with the TLC results. Especially for ClWS / DGAT2-3, the fatty acid content was low and the TLC detection bands were not obvious.

[0034] In summary, it is of great significance to analyze the effects of different DGAT genes on the oil yield of Coix lacryma-jobi by using transgenic technology and molecular biology methods in the present invention. The present invention mainly carried out the cDNA cloning of the Coix lacryma-jobi DGAT gene family and analyzed the oil production rate of yeast using the defective H1346 yeast, laying a foundation for further exploring the molecular mechanism of Coix lacryma-jobi oil synthesis. Brief Description of the Drawings

[0035] The following further elaborates on the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0036] Figure 1 It is the amplification results of 10 cDNA; the lanes from left to right respectively correspond to the amplification product bands of DL 2000 Marker, ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, and ClWS / DGAT2-4 gene fragments.

[0037] Figure 2 It is the PCR of 10 recovered plasmids; the lanes from left to right respectively correspond to Marker and the plasmid product bands with ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, and ClWS / DGAT2-4 fragments.

[0038] Figure 3 It is the yeast colony PCR; the lanes from left to right respectively correspond to Marker and the yeast colony PCR product bands of plasmids respectively ligated with ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, and ClWS / DGAT2-4 fragments.

[0039] Figure 4 It is the bar graph of the total fatty acid content in yeast.

[0040] Figure 5 It is the bar graph of the fat content in yeast.

[0041] Figure 6 It is the TLC result diagram.

[0042] Figure 7 It is the Nile red staining of the oils of ten positive yeasts. Specific Embodiments

[0043] The following further describes the present invention in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0044] Example 1: Gene Cloning of the DAGT Gene Family in Coix lacryma-jobi

[0045] Experimental Materials:

[0046] Plant materials: The plant materials were taken from the Coix germplasm resource nursery of the School of Life Sciences and Medicine, Zhejiang Sci-Tech University. A Coix plant with good growth was selected, and the Coix fruits were picked for the extraction of total RNA.

[0047] Reagent materials

[0048] Product Name Manufacturer TIANGEN DP432 RNAprep Pure Total RNA Extraction Kit for Plants TianGEN 2×Phanta FLAsh Master Mix (Dye plus) P520 Vazyme Reverse Transcription Kit Vazyme UE-GX-250 DNA Gel Extraction Kit Suzhou Uyland Biotech Agarose Biowest β-Mercaptoethanol Macklin

[0049] Specifically as follows:

[0050] 1), Identification of Coix DGAT gene sequences

[0051] Search for the CDS sequences of DGAT genes of Arabidopsis thaliana, Zea mays, and Sorghum bicolor in NCBI, and use the homologous comparison method to screen the Coix genome by Blast. Finally, 10 Coix DGAT genes were identified in four subfamilies of DGAT1, DGAT2, DGAT3, and WS / DGAT2. According to the classification, they were named ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, and ClWS / DGAT2-4. Specifically as shown in Table 1 below.

[0052] Table 1 Identification table of Coix DGAT genes

[0053] ID Length (bp) Corresponding Number in Sequence Listing Nomenclature Subfamily Cl027022_T1 1383 SEQ ID NO:1 ClDGAT1-1 DGAT1 Cl031330_T1 1233 SEQ ID NO:2 ClDGAT1-2 DGAT1 Cl042438_T3 1344 SEQ ID NO:3 ClDGAT1-3 DGAT1 Cl018010_T2 1473 SEQ ID NO:4 ClDGAT2-1 DGAT2 Cl027481_T3 933 SEQ ID NO:5 ClDGAT2-2 DGAT2 Cl031870_T1 1260 SEQ ID NO:6 ClDGAT3 DGAT3 Cl002154_T2 678 SEQ ID NO:7 ClWS / DGAT2-1 WS / DGAT2 Cl011297_T1 1506 SEQ ID NO:8 ClWS / DGAT2-2 WS / DGAT2 Cl013404_T1 1566 SEQ ID NO:9 ClWS / DGAT2-3 WS / DGAT2 Cl041689_T1 1632 SEQ ID NO:10 ClWS / DGAT2-4 WS / DGAT2

[0054] 2), Extraction of total RNA from Coix

[0055] RNA was extracted using the Tiangen DP432 RNAprep Pure Plant Total RNA Extraction Kit. The RNA was reverse transcribed into cDNA using an RNA reverse transcription kit.

[0056] The obtained RNA solution was detected using NanoDrop, and the concentration was 306.8 ng / μl. Part of it was taken out and reverse transcribed into cDNA, and the rest was stored at -80 °C for later use. The obtained cDNA solution was detected for concentration using NanoDrop, and the concentration was 32.4 ng / μl, and it was stored at -4 °C for later use.

[0057] 3), Cloning of DGAT genes

[0058] ①, Primer design

[0059] All primers in Table 2 were designed using CE Design software. The primer names and sequences are shown in Table 2:

[0060] Table 2 Cloning primers for ten DGAT genes

[0061] Gene Name Primer Name Primer Sequence ClDGAT1-1 DGAT1_1-F cttggtaccgagctcggatccATGGCCCCGCCCCCCTCC DGAT1_1-R gcggccgttactagtggatccCTATCTGCTTGCCTGGGCC ClDGAT1-2 DGAT1_2-F cttggtaccgagctcggatccATGGCGGACTCCGACGAC DGAT1_2-R gcggccgttactagtggatccTTATTTTGTCTTCTCAGTCCGGTTC ClDGAT1-3 DGAT1_3-F cttggtaccgagctcggatccATGAAACGAGACGAGACCGACA DGAT1_3-R gcggccgttactagtggatccTCATGTGCCACCATTATATTCTGG ClDGAT2-1 DGAT2_1-F cttggtaccgagctcggatccATGCAGAAACCTGTCTCACACG DGAT2_1-R gcggccgttactagtggatccTCAAAGAACTCTTAACCGAAGATCAG ClDGAT2-2 DGAT1_1-F cttggtaccgagctcggatccATGGCCCCGCCCCCCTCC DGAT1_1-R gcggccgttactagtggatccCTATCTGCTTGCCTGGGCC ClDGAT3 DGAT3-F cttggtaccgagctcggatccATGGAGCTCACCGGCGCC DGAT3-R gcggccgttactagtggatccCTAGATAGGCGTCATGCCAAGAT ClWS / DGAT2-1 WSDGAT2_1-F cttggtaccgagctcggatccATGGCGGTCGCTGCCCAA WSDGAT2_1-R gcggccgttactagtggatccTTAAACCTTGCTGATGTTATTAGTTGTATC ClWS / DGAT2-2 WSDGAT2_2-F cttggtaccgagctcggatccATGCATGCAACCAGCCATCT WSDGAT2_2-R gcggccgttactagtggatccTTAATCGTTTTTGGTTGACGCC ClWS / DGAT2-3 WSDGAT2_3-F cttggtaccgagctcggatccATGGATGCGGCGGCAGCC WSDGAT2_3-R gcggccgttactagtggatccTTAATCATTTCTTTGATTCTTTTCAATG ClWS / DGAT2-4 DGAT2_1-F cttggtaccgagctcggatccATGCAGAAACCTGTCTCACACG DGAT2_1-R gcggccgttactagtggatccTCAAAGAACTCTTAACCGAAGATCAG

[0062] ②. Cloning of ten DGAT genes: Configure the following PCR buffer system:

[0063]

[0064] Use the following PCR reaction program for PCR amplification:

[0065]

[0066]

[0067] Set the temperature gradient to 63°C - 76°C and 50°C - 63°C for PCR reaction around the annealing temperature automatically generated during primer design, so as to determine the optimal annealing temperature (Tm) of the ten DGAT genes. Specifically as shown in Table 3 below:

[0068] Table 3 Optimal Tm values for PCR amplification of Coix DGAT genes

[0069] ID Gene name Tm (°C) Cl027022_T1 ClDGAT1_1 63 Cl031330_T1 ClDGAT1_2 63 Cl042438_T3 ClDGAT1_3 63 Cl018010_T2 ClDGAT2_1 76 Cl027481_T3 ClDGAT2_2 63 Cl031870_T1 ClDGAT3 63 Cl002154_T2 ClWS / DGAT2_1 76 Cl011297_T1 ClWS / DGAT2_2 73.5 Cl013404_T1 ClWS / DGAT2_3 60 Cl041689_T1 ClWS / DGAT2_4 76

[0070] ③. Gel cutting, recovery and purification of DGAT gene fragments

[0071] Perform PCR reaction on the 10 DGAT genes according to the above PCR program and the corresponding optimal annealing temperature (Tm). After obtaining the PCR products, perform 1% agarose gel electrophoresis at 120V for 30 min (all electrophoresis in this invention uses this condition), use the 2000bp Marker as a control, and perform gel cutting and recovery after the target band position is correct. And perform corresponding operations in the conventional manner to obtain a purified DNA solution.

[0072] The results of the gel diagram of the amplification products are as Figure 1 shown. After gel cutting and recovery of the target fragment, perform concentration detection on the NanoDrop, and the obtained Oligo concentrations are as shown in Table 4 below:

[0073] Table 4 Concentrations of recovered fragments of Coix DGAT genes

[0074]

[0075]

[0076] The concentrations of the cDNA fragment recovery solutions are all within the concentration range available for vector construction. Among them, the recovery concentration of the ClWS / DGAT2-2 cDNA fragment is relatively low, and the construction system can be doubled in the subsequent expression vector construction.

[0077] Example 2. Construction of expression vector

[0078] The main reagents are as follows:

[0079]

[0080] Main culture medium

[0081] LB liquid medium: Weigh 5 g of yeast powder, 10 g of Tyrptone, and 10 g of NaCl, dissolve them in 1000 mL of ddH2O, and sterilize them in an autoclave at 121 °C for 15 min.

[0082] LB solid medium with Amp resistance: First add 20 g / L of agar powder to the LB liquid medium and then sterilize it. After taking it out, inject 0.5 ml of 100 mg / ml Amp solution into a 0.22-μm microporous membrane filter through a sterile syringe, filter it, and add it to the medium that has not solidified and has cooled to about 60 °C, and mix well.

[0083] Specifically as follows:

[0084] 1), Digestion of pYes2.0 plasmid

[0085] Use BanHI-HF restriction endonuclease to perform single digestion on the pYes2 plasmid. Prepare the following system on ice:

[0086]

[0087] Gently pipette and mix well, and perform the reaction in a PCR instrument at 37 °C for 15 min to obtain the linearized cloning vector of pYes2.0 plasmid.

[0088] Measure the obtained linearized cloning vector of pYes2.0 plasmid using NanoDrop: The solution concentration is 22 ng / μl.

[0089] 2), Ligation of gene fragments

[0090] Use the ClonExpress Ultra ONE Step Cloning Kit C115 seamless cloning kit to perform the ligation of gene fragments. The usage amount of each fragment is calculated according to the formula:

[0091] Optimal usage amount of cloning vector = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0092] Optimal usage amount of inserted fragment = [0.04 × number of base pairs of inserted fragment] ng (0.06 pmol)

[0093] Prepare the ligation reaction system for 10 genes on ice according to the following system:

[0094]

[0095] Gently pipette to mix well and centrifuge briefly. Heat in a metal bath at 50 °C for 5 min, and immediately cool on ice; thus, 10 recombinant vectors are obtained correspondingly.

[0096] 3) Transformation of the ligation product into DH5α Escherichia coli competent cells

[0097] Thaw the DH5α Escherichia coli competent cells on ice. Pipette 5 μl from each of the 10 recombinant vectors and add them separately to 50 μl of the competent cells. Flick the tube wall gently to mix well, and let it stand on ice for 30 min. Heat shock in a metal bath at 42 °C for 45 sec, and immediately cool on ice for 3 min. Add 1 ml of LB liquid medium to each centrifuge tube, and place it in a shaker at 37 °C and 200 rpm for 40 min. In a laminar flow hood, pipette 100 μl of the bacterial liquid and spread it evenly on an Amp-resistant LB plate with a sterile glass bead. Incubate the plate upside down in an incubator at 37 °C for 14 h.

[0098] 4) Screening of positive Escherichia coli colonies

[0099] Use a pipette tip to pick a single Escherichia coli colony and resuspend it in 100 μl of sterile water for colony PCR. The PCR uses the pYes2.0 universal primers, and the primer names and sequences are shown in Table 5:

[0100] Table 5 pYes2.0 plasmid universal primer sequences

[0101]

[0102] Prepare the reaction system according to the following table:

[0103]

[0104] Use the following PCR reaction program

[0105]

[0106]

[0107] Reserve the positive Escherichia coli bacterial liquid showing the correct band for future use.

[0108] 5) Scale-up culture

[0109] Transfer the obtained positive Escherichia coli bacterial liquid to a 50-ml centrifuge tube containing 20 ml of LB liquid medium, and place it in a shaker at 37 °C and 200 rmp for 14 h.

[0110] 6) Plasmid preparation

[0111] ① Plasmid extraction

[0112] Use the UE-MN-P-50UE plasmid miniprep kit to extract plasmids from the Escherichia coli broth after expanded culture. Add RNaseA and absolute ethanol to the lysis buffer S1 and Buffer W2 respectively according to the instructions in advance. Take out the centrifuge tube from the incubator, centrifuge at 5000 rpm for 15 min, and discard the supernatant. Resuspend the cells by adding 750 μl of lysis buffer S1 and S2 and 1000 μl of lysis buffer S3, and mix well by oscillation. Aliquot the mixture into 1.5 ml centrifuge tubes and centrifuge at 12000 rpm for 10 min. Transfer the supernatant to the preparation tube, centrifuge at 12000 rpm for 1 min, discard the filtrate, and put the preparation tube back into the collection tube. Repeat the above process until all the supernatant has been filtered through the preparation membrane. Add 500 μl of Buffer W1 reagent to the preparation tube, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 700 μl of Buffer W2, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat the washing once. Discard the filtrate and centrifuge at 12000 rpm for 1 min. Place the preparation tube on a clean centrifuge tube, and use a pipette to add 200 μl of Eluent dropwise to the center of the preparation membrane. Let it stand for 15 min and centrifuge at 12000 rpm for 1 min to obtain the plasmid solution.

[0113] The concentration of the obtained recombinant plasmid vector (plasmid solution) was detected on a NanoDrop protein and nucleic acid analyzer, and the measured concentration range was between 52.4 and 149.5 ng / μl (Table 6). The concentration of each plasmid required in this experiment needs to be above 30 ng / μl, and the concentrations of the 10 recombinant plasmids are all above the required concentration and can be used for yeast transformation.

[0114] Table 6 Concentrations of 10 recovered recombinant plasmids

[0115] Plasmid DNA Concentration (ng / μl) ClDGAT1_1 71.6 ClDGAT1_2 109.0 ClDGAT1_3 92.1 ClDGAT2_1 77.0 ClDGAT2_2 52.4 ClDGAT3 149.5 ClWS / DGAT2_1 62.5 ClWS / DGAT2_2 73.4 ClWS / DGAT2_3 76.2 ClWS / DGAT2_4 124

[0116] ②. Plasmid verification

[0117] The plasmid solution obtained in the present invention was also verified by PCR. Prepare a 1.5% agarose gel, perform electrophoresis using DL2000 Marker and the obtained PCR products, and observe whether the product bands conform to the corresponding positions in the ultraviolet gel imaging system.

[0118] The obtained results were as follows: The obtained plasmid was subjected to agarose gel electrophoresis, and it was found that the main band was very bright ( Figure 2 ), and the size was consistent with that shown in Table 2, indicating that the target band had been successfully ligated to the vector.

[0119] In summary, 10 recombinant plasmids of DGAT genes were obtained.

[0120] Example 3. Transformation of 10 recombinant plasmids of DGAT genes into H1246 yeast

[0121]

[0122] Main culture medium

[0123] YPDA liquid medium: Weigh 50 g of YPDA powder produced by Coolaber and dissolve it completely in 1000 ml of ddH2O. Put it into an autoclave and sterilize it at 121 °C for 15 min.

[0124] YPDA solid medium: Add 20 g / L of agar powder additionally before sterilization.

[0125] SC-URA inhibitory medium: Dissolve 8 g of SC-URA, 20 g of glucose in 1000 mL of ddH2O, stir well to dissolve, and then put it into an autoclave and sterilize it at 115 °C for 20 min.

[0126] SC-URA solid inhibitory medium: Add 20 g / L of agar powder additionally before sterilization.

[0127] SC-URA induction medium: Dissolve 8 g of SC-URA in 900 mL of ddH2O, put it into an autoclave and sterilize it at 115 °C for 20 min. Dissolve 10 g of galactose and 10 g of raffinose in 100 ml of ddH2O, heat it to 70 °C in a water bath until completely dissolved, and then filter it through a 0.22-μm microporous filter with a sterile syringe and add it to the above-mentioned SC-URA medium that has been sterilized by high temperature.

[0128] Specifically as follows:

[0129] 1), Preparation of H1246 yeast competent cells

[0130] Thaw the H1246 yeast cells stored at -80 °C on ice and prepare competent cells in a conventional manner.

[0131] 2), Transformation of plasmid into H1246 yeast competent cells

[0132] Prepare the plasmid transformation premix according to the following table:

[0133]

[0134]

[0135] Transform H1246 yeast competent cells:

[0136] Add 1 tube of premixed solution to each of the 10 tubes of H1246 competent cells, pipette up and down repeatedly to thoroughly suspend the yeast cells. Incubate in a metal bath at 30°C for 30 min, mixing every 10 min. After that, heat shock in a metal bath at 42°C for 30 min, mixing every 10 min. After completion, centrifuge at 12,000 rpm for 15 s and discard the supernatant. Resuspend each tube with 1 mL of YPDA liquid medium and culture at 30°C and 200 rpm for 40 min. Take 5 μL of the bacterial solution from each tube and add it to 200 μL of sterile water. Take 100 μL of the diluted bacterial solution and spread it on a SC-URA inhibitory medium plate with glass beads, and incubate it upside down at 30°C for 3 days.

[0137] 3) Screening for positive H1246 yeast

[0138] ① Yeast cell wall breaking

[0139] Pick well-grown yeast single colonies from the medium in a laminar flow hood and resuspend them in 100 μL of sterile water. Prepare the wall-breaking system according to the following table:

[0140]

[0141] Break the cell wall according to the following procedure: Immediately place it in liquid nitrogen for 5 min after 10 min at 98°C, repeat 2 times, and finally take it out after another 10 min at 98°C. Obtain the wall-broken yeast cell bacterial solution.

[0142] ② Colony PCR

[0143] Prepare the PCR system according to the following table:

[0144]

[0145] Perform PCR according to the following procedure:

[0146]

[0147] Colonies with matching bands are positive colonies, and expand the culture using 1 ml of SD-URA liquid medium. After 2 days, mix 500 μL of the bacterial strain with 500 μL of 60% glycerol and store it in a -80°C refrigerator. The remaining bacterial solution is used for subsequent experiments.

[0148] The results obtained are as follows:

[0149] Perform agarose gel electrophoresis on the obtained PCR products and find that some of the main bands are very bright and some are relatively dim ( Figure 3 ), but all are in line with the size shown in Table 1, indicating that the picked colonies contain the target recombinant vector and the target vector has been successfully transformed into H1246 yeast.

[0150] As Figure 3As shown: Due to different bacterial concentrations, the brightness of the bands varies. The ClWS / DGAT2-2 band has two brighter bands. The lower band is consistent with the size of the band, and the upper band may be a miscellaneous band caused by poor primer specificity.

[0151] 4) Expand training

[0152] ① Pre-cultivation

[0153] The positive yeast culture was transferred to a 1.5 ml centrifuge tube containing 1 ml SC-URA inhibition culture medium and cultured at 30°C, 200 rpm for 2 days for expansion culture.

[0154] ②, Culture medium preparation

[0155] Prepare 190 ml of SC-URA liquid culture medium in 10 conical flasks and sterilize them by autoclaving at 115°C for 20 min. Dissolve 20 g of raffinose and 20 g of galactose in 100 ml of ddH2O and heat in a water bath, stirring to dissolve completely. After the 10 bottles of liquid culture medium are sterilized, use a 20 ml sterile syringe and filter in a clean bench to add 10 ml of mixed sugar solution to each conical flask to complete the preparation of 10 bottles of SC-URA liquid induction medium (1% galactose, 1% raffinose), and keep them for later use.

[0156] ③. Expand training

[0157] 900 μl of the obtained pre-culture medium was inoculated into a conical culture flask containing 200 ml of liquid SC-URA induction medium (1% galactose, 1% raffinose) prepared in 4.2.4.2, and cultured in a shaking incubator at 30°C and 200 rpm for 5 days for yeast oil extraction.

[0158] Expanded culture results

[0159] OD of 10 bottles of bacterial solution after 5 days of continuous culture 600 The values were all above 1, and there was no contamination. The culture was successfully expanded and can be used for subsequent experiments.

[0160] Example 4, Yeast Oil Extraction

[0161] Experimental Materials

[0162] Cell material: The bacterial solution obtained by expanding the positive yeast obtained in Example 3 for 5 days.

[0163] Main reagents: T6221-23-20EA thin layer chromatography silica gel plate i-Quip

[0164] The details are as follows:

[0165] 1) Preparation of yeast powder

[0166] Take 1 ml of the bacterial liquid from each of the 10 bottles of culture medium for Nile red staining, and prepare yeast powder from the rest. Transfer the remaining parts of the 10 bottles of culture medium to 10 centrifuge tubes of 50 ml respectively, and centrifuge at 4000 rpm for 5 min. Discard the supernatant, pour in the culture medium again, and continue centrifuging until all the culture medium in the conical flask has been centrifuged. Put the obtained yeast cell precipitate into a freeze dryer and dry it for 24 h before taking it out. Obtain dry solid yeast blocks and weigh the total mass.

[0167] Specifically as shown in Table 7

[0168] Table 7 Total mass of yeast powder

[0169]

[0170]

[0171] The solid yeast powder obtained by freeze-drying the precipitate obtained by centrifugation has a mass between 244 mg and 511 mg, meeting the requirement of 210 mg for the experiment.

[0172] 2) Extraction of total yeast fatty acids

[0173] Weigh 3 portions of 50 mg samples from each of the obtained 10 positive yeast powders and place them into 1.5 ml centrifuge tubes. Add three sterilized small steel beads to each centrifuge tube. Subsequently, use a grinder for grinding, grinding twice at 60 HZ for 60 sec. Take out the centrifuge tubes and perform subsequent operations in a fume hood. Add 1 ml of chloroform to each tube and pipette repeatedly until the powder is completely suspended in the solution. Transfer the solution to a 50 ml centrifuge tube. Prepare 300 ml of chloroform-methanol (v:v = 1:2) mixture in a beaker. Add 7.5 ml of chloroform-methanol mixture to each 50 ml centrifuge tube, mix well, and place it on a shaker at 37 °C and 200 rpm for 24 h. Centrifuge at 4000 rpm for 5 min and collect the supernatant into corresponding 50 ml centrifuge tubes. Then add 7.5 ml of chloroform-methanol (v:v = 1:2) mixture to the original centrifuge tube, place it on a shaker at 37 °C and 200 rpm, and take it out after 12 h. Centrifuge at 4000 rpm for 5 min and collect the supernatant into corresponding 50 ml centrifuge tubes. Then add 7.5 ml of chloroform-methanol (v:v = 1:2) mixture to the original centrifuge tube, place it on a shaker at 37 °C and 200 rpm for 12 h. Centrifuge at 4000 rpm for 5 min and collect the supernatant. Prepare 400 ml of 1% NaCl solution. Add 10 ml of 1% NaCl solution and 5 ml of chloroform to each of the 30 tubes of supernatant, shake well, and centrifuge at 4000 rpm for 5 min. Transfer the lower chloroform phase to a pre-weighed 15 ml centrifuge tube, and do not aspirate the upper solution. Heat the 15 ml centrifuge tube in a water bath until the organic phase evaporates, then place it in an oven to dry, cool to room temperature, and weigh and calculate the total fatty acid content.

[0174] Total fatty acid content = (total mass after extraction - weight of centrifuge tube before extraction) / yeast mass

[0175] The results of total fatty acid extraction from yeast are as follows:

[0176] As Figure 4 shown, the fatty acid contents extracted from 50 mg yeast powders of 10 positive yeasts are shown. From left to right, they are ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, ClWS / DGAT2-4. The contents are between 20.00% and 36.27%, and the extraction rates are all relatively high. However, different DGAT genes have different efficiencies in restoring oil production in H1246 yeast. Among them, the yeast strains inserted with ClDGAT2-2, ClDGAT3, and ClWS / DGAT2-4 fragments have relatively high fatty acid contents; the yeast strains inserted with ClDGAT1-1, ClDGAT2-1, and ClWS / DGAT2-3 gene fragments have relatively low fatty acid contents.

[0177] 3) Total fat extraction of yeast

[0178] Weigh 3 portions of 20 mg samples from each of the 10 positive yeast powders obtained and place them into 1.5 ml centrifuge tubes whose weights have been measured, and grind them according to the steps in the previous step. Prepare 30 ml of chloroform: methanol solution (v:v = 2:1) and 10 ml of 0.9% (w / v) potassium chloride solution. After grinding, add 1 mL of chloroform: methanol solution (v:v = 2:1) to each centrifuge tube, repeatedly blow and suck to resuspend the powder, let it stand for 1 min, aspirate the mixture into a 4 ml centrifuge tube, add 0.5 ml of 0.9% (w / v) potassium chloride solution and 1 ml of chloroform: methanol solution with a pipette, shake and mix well, centrifuge at 4000 rpm for 5 min and then take out. It can be seen that the mixture in the centrifuge tube becomes very clear and there is an obvious layering phenomenon. The upper layer should be methanol - potassium chloride solution, the lower layer is the chloroform phase, and the middle thin layer should be cell residue; aspirate the lower chloroform phase and transfer it to a new 1.5 ml centrifuge tube, heat it in a water bath to evaporate the organic phase completely, put it in an oven and dry it at 45 °C for 3 h, then weigh the total weight and calculate the total fat extraction rate of yeast.

[0179] Total fat content = (total mass after extraction - weight of centrifuge tube before extraction) / yeast mass

[0180] After weighing, store it at -4 °C for later use.

[0181] The results of total fat extraction of yeast are as follows:

[0182] As Figure 5 shown, it is the fat content extracted from 20 mg yeast powder of 10 positive yeasts. From left to right, they are ClDGAT1 - 1, ClDGAT1 - 2, ClDGAT1 - 3, ClDGAT2 - 1, ClDGAT2 - 2, ClDGAT3, ClWS / DGAT2 - 1, ClWS / DGAT2 - 2, ClWS / DGAT2 - 3, ClWS / DGAT2 - 4. The total fat content is between 12.17% and 33.83%. Among them, the yeast strains transfected with the gene fragments of ClDGAT1 - 2, ClDGAT2 - 2 and ClDGAT3 have relatively high fat production; the yeast strains transfected with the gene fragments of ClDGAT1 - 3, ClDGAT2 - 1 and ClWS / DGAT2 - 4 have relatively low fat production.

[0183] 4) TLC chromatography of yeast

[0184] Prepare 100 μl of standard (50 μl of triglyceride + 50 μl of diglyceride) and 101 ml of developing solvent n - hexane:ether:formic acid (v:v:v = 80 mL:20 mL:1 mL). Take out two silica gel plates, divide each sample area into 1.5 cm wide, the height of the spotting point is 1 cm from the lower edge. Take one tube of each of the yeast fat samples prepared above, add 100 μl of chloroform solution, and blow and suck repeatedly to completely dissolve the oil. Use a brand - new capillary tube to aspirate a total of 10 μl of each sample for spotting; add an appropriate amount of developing solvent to the chromatography tank, place the silica gel plate into the chromatography tank with the spotting side facing down and the sample not immersed in the liquid, and take out the silica gel plate before the developing solvent reaches the upper edge of the silica gel plate. Increase the exhaust intensity of the fan in the fume hood and place the silica gel plate in it for drying; place the dried silica gel plate in an iodine tank, cover the lid, and stain with the spotting side facing up for 30 min or longer to better stain the diffused oil. Take it out when the oil trace is very obvious and immediately take a photo for recording.

[0185] The TLC chromatographic analysis of yeast is as follows:

[0186] The TLC chromatographic results are as Figure 6 shown. The first spotting position on each silica gel plate is the standard of equimolar mixture of triglyceride and diglyceride, and then from left to right are ClDGAT3, ClWS / DGAT2 - 1, ClWS / DGAT2 - 2, ClWS / DGAT2 - 3, ClWS / DGAT2 - 4, ClDGAT1 - 1, ClDGAT1 - 2, ClDGAT1 - 3, ClDGAT2 - 1, ClDGAT2 - 2, 20 μl each. Except for the faint band of ClWS / DGAT2 - 3, all samples have obvious bands at triglyceride, indicating that the expression of these 10 genes has successfully restored the ability of yeast to synthesize TAG, and the proteins encoded by these genes have the function of DGAT enzyme.

[0187] 5) Nile red staining of yeast

[0188] Prepare the Nile red staining system in the dark:

[0189]

[0190] After preparation, mix well

[0191] Centrifuge the 10 tubes of positive yeast solution retained above at 8000rpm for 5 minutes and discard the supernatant. Resuspend the bacteria with 1ml sterile water, centrifuge at 8000rpm for 5 minutes, discard the supernatant, and repeat the washing 3 times. Finally, resuspend with 1ml sterile water, and take 100μl of the bacterial solution into new sterile centrifuge tubes for use. Stain the bacterial solution in a dark place, use a pipette to draw 10μl of Nile red dye and add it to 10 tubes of 100μl bacterial solution and immediately shake vigorously until the bacterial solution presents a uniform, transparent light red color, and stand in the dark for 10 minutes. Gently wipe the slide and glass slide with lint-free paper, draw 10μl of bacterial solution from each tube to make a glass slide, and observe the results using a fluorescent inverted microscope.

[0192] Nile red staining analysis of yeast is as follows:

[0193] like Figure 7 As shown, the positive yeast containing ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, and ClWS / DGAT2-4 connected respectively were stained with oil (gold) in the detection results of the fluorescent inverted microscope after Nile red staining, indicating that each gene restored the ability to produce oil after transforming the defective H1246 yeast. In summary, it shows that the plasmid transformation is successful.

[0194] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of Coix lacryma-jobi diacylglycerol acyltransferase gene ClDGAT2 in increasing oil yield.

2. The application according to claim 1, wherein: It can produce triglycerides.

3. The application according to claim 1 or 2, characterized in that: It has the function of DGAT enzyme.

4. The application according to claim 3, wherein: It has the ability to restore oil production in TAG-deficient Saccharomyces cerevisiae.

5. The application according to any one of claims 1 to 4, characterized in that: The Coix lacryma-jobi diacylglycerol acyltransferase gene ClDGAT2 is respectively the following 10 genes: ClDGAT1-1, ClDGAT1-2, ClDGAT1-3, ClDGAT2-1, ClDGAT2-2, ClDGAT3, ClWS / DGAT2-1, ClWS / DGAT2-2, ClWS / DGAT2-3, ClWS / DGAT2-4; The corresponding sequences are shown as no1 to no10 in the sequence listing.

6. The application according to claim 5, characterized in that: 1), Each Coix lacryma-jobi diacylglycerol acyltransferase gene ClDGAT2 is respectively operated as follows: ligated to the pYes2 vector and transformed into DH5α Escherichia coli competent cells. After PCR, the positive bacteria with the correct band position are expanded in culture, and the plasmid is extracted to obtain the corresponding ClDGAT2 gene recombinant plasmid; 2), The ClDGAT gene recombinant plasmid is transformed into H1246; it is cultured with SC-Ura solid inhibitory medium. After screening, the obtained positive yeast cells containing the ClDGAT gene recombinant plasmid are expanded in culture to obtain a bacterial solution for oil extraction.

7. The application according to claim 6, characterized in that: The yeast strains transfected with the ClDGAT2-2, ClDGAT3 and ClWS / DGAT2-4 gene fragments produce a high fatty acid content; The yeast strains transfected with the ClDGAT1-2, ClDGAT2-2 and ClDGAT3 gene fragments produce a high fat content.

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