CN106319022B - Screening and identifying method of 3-phosphoglycerol acyltransferase gene - Google Patents
Screening and identifying method of 3-phosphoglycerol acyltransferase gene Download PDFInfo
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Abstract
The invention relates to a screening and identifying method of 3-phosphoglycerol acyltransferase gene, which constructs condition lethal yeast double mutant ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]), establishes a rapid and high-efficiency in vivo genetic complementary screening system, is applied to screening GAPTs genes containing or not containing acyltransferase characteristic sequences in eukaryotes, and can also be applied to screening anti-obesity drugs in medicine.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to the separation and identification of a 3-phosphoglyceryl acyltransferase (GPATs) gene and a gene participating in a first-step acylation reaction enzyme in a glyceride synthesis pathway in a eukaryotic cell; screening GPATs enzyme inhibitors; and optimization and modification of GPATs gene sequences.
Background
In eukaryotes, the initial and second acylation reactions of de novo biosynthesis of glycerolipids are catalyzed by 3-phosphoglycerate acyltransferases (GPATs) and lysophosphatidic acid acyltransferases (LPATs), respectively. At present, it is still unclear how many genes encoding GPATs and LPATs are contained in higher plants and mammals.
With the development and application of large-scale sequencing technology, it is found that many gene-encoded proteins contain conserved acyltransferase characteristic sequences. However, most membrane-bound GPATs and LPATs contain these signature sequences, and thus it is difficult to distinguish which class of acyltransferase these genes encode by bioinformatic analysis (Lewis et al, 1999; Zheng et al, 2003). In addition, it is still unclear whether there is a class of GPATs in eukaryotes that do not contain acyltransferase signature sequences. Therefore, in order to effectively analyze the first step of key reaction in the glycerolipid synthesis pathway, an efficient GPATs screening and identifying system needs to be established.
GPATs enzymatic activity is characterized primarily by in vitro enzymatic analysis and in vivo genetic function complementation experiments (Zheng and Zou, 2001; McIntyre et al, 1977; Lewis et al, 1999; Lindner et al, 2014). In vitro enzymatic analysis relies on radioisotope labeling and is not suitable for high throughput screening; the genetic function complementation test is considered as an effective screening method in vivo. In prokaryotes, the reported Escherichia coli mutant plsB is a GPATs functional deficient strain and is suitable for screening GPATs genes of prokaryotes. However, the main acyl donor of the prokaryotic type II fatty acid synthesis pathway is the acyl carrier protein (acyl-ACP), which is distinguished from the eukaryotic acyl donor acyl-CoA (acyl-CoA), making this system inapplicable to the screening of eukaryotic GPATs (Murata et al, 1997; Zheng and Zou, 2001; Zhang and Rock, 2008; Manas-Fernandez et al, 2010; Lindner et al, 2014).
Glycerol synthesis in eukaryotes is highly conserved, and thus, a highly efficient and specific GPATs genetic complementation screening system can be established using yeast GPATs function-deficient mutants (Wilkison and Bell, 1997; Athenstaedtand Daum, 1999; Zheng and Zou, 2001; Zheng et al, 2003). Saccharomyces cerevisiae contains two genes encoding 3-phosphoglyceromyceroytransferase GAT1 and GAT2 at the sn-1 position, catalyzing the first acylation reaction of the glycerol biosynthesis pathway. studies show that simultaneous knock-out of these two genes makes yeast double mutants lethal (Zheng and Zou, 2001; Zarmemberg and McMaser, 2002). in the genetic background of the W303 strain, two conditional double knock-out mutants (GAT1 △ GAT2 △ + [ 829 4:: GAT) 3 and 58 VZY23(GAT 2 △: [ GAT 387 5 URA3]) and pG 3 can grow in normal medium containing galactose (pGbert 363623; pGbert 2; pGbert 2 △: GAT2 △ +: GAT 2).
We constructed two conditional double knockout mutants of BY-NIU8(BY4742, GAT1 △ GAT2 △ + [ pGAL1:: GAT1URA 3]) and BY-LEI5(BY4742, GAT1 △ GAT2 △ + [ pGAL1:: GAT2URA3 ]). the results showed that both mutants grew in media containing galactose and glucose (GAL1 is a galactose-inducible and glucose-repressible promoter). We speculated that it was possible that yeast-own GAT1 and GAT2 complementary genes were expressed at low levels in media containing glucose, thereby maintaining growth of yeast double mutants, which would not be suitable for candidate GPATs screening, indicating that the conditional double mutants would need further optimization and modification.
It is generally accepted that the catalytic activity of an enzyme is higher in the bulk cells and lower in the heterologous cells. We attempted to replace the yeast GAT1 and GAT2 genes with a heterologous complementing gene of low catalytic activity, and constructed conditional double mutants that could restore growth in media containing galactose due to high expression of the heterologous complementing gene or other genes encoding sn-1 acyltransferase, but not enough to restore growth in media containing glucose with low expression. Previous findings indicate that AtGPAT1 and AtGPAT5 in the Arabidopsis AtGPATs gene family have similar gene functions as Gat1 and Gat2, but the enzymatic activity of heterologous expression in yeast cells is 10-fold lower than that of yeast's own GPATs (Gat1p and Gat2p) (Zheng and Zou, 2001; Zheng et al, 2003). Based on the hypothesis and the research result, a novel, specific and stable yeast genetic complementation system is constructed by using the characteristics of AtGPAT1 and AtGPAT5 and is used for screening eukaryotic GPATs, and the yeast genetic complementation screening system can quickly and effectively identify an acyltransferase gene and promote the research of a glycerophospholipid de novo biosynthesis pathway.
Disclosure of Invention
The invention constructs conditional lethal yeast double mutant ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]), establishes a rapid and efficient in vivo genetic complementation screening system, is applied to screening of GAPTs genes containing or not containing acyltransferase characteristic sequences in eukaryotes, and can also be applied to screening of anti-obesity drugs in medicine.
The invention provides a method for screening and identifying 3-phosphoglyceryl transferase (GPATs) genes, which comprises the following steps:
step one, constructing an intermediate yeast double mutant: introducing an expression vector with a heterologous GPATs gene into a yeast double mutant of an expression vector with yeast self GAT1 or GAT2 gene (encoding GPATs) by a yeast genetic transformation method to obtain an intermediate yeast double mutant containing two expression vectors with a yeast GAT1 (or GAT2) gene and a heterologous GPAT gene respectively;
step two, shaking the intermediate yeast double mutant obtained in the step one through a uracil-Urea (URA) -containing liquid culture medium overnight, and then screening on a solid culture medium containing 5-fluoroorotic acid (5-FOA) and URA to obtain a new conditional lethal yeast double mutant of the expression vector containing a heterologous GPAT gene;
and step three, constructing a candidate GPATs gene expression vector by using the modified pYES2-yADH1-Kan novel plasmid, and introducing the candidate GPATs gene expression vector into a new conditional lethal yeast double mutant.
And step four, adopting a culture medium containing glucose as a screening condition, and enabling the new conditional lethal yeast double mutant to recover the growth of a heterologous gene in the glucose culture medium, namely the gene encoding the GPAT enzyme activity.
Preferably, the heterologous GPATs genes in the first step of the screening and identifying method of GPATs genes are AtGPAT1 and AtGPAT5 in Arabidopsis thaliana.
Preferably, the protein sequence coded by the heterologous GPATs gene in the step one of the screening and identifying methods of the GPATs gene has homology of more than 85% with the protein sequences of AtGPAT1 and AtGPAT5 in Arabidopsis thaliana.
Preferably, the genes having homology of more than 85% with the AtGPAT1 and AtGPAT5 protein sequences in Arabidopsis thaliana in the screening and identifying method of GPATs genes include Arabidopsis thaliana (Arabidopsis lyrata), Camelina sativa (Camelina sativa), shepherd's purse (Capsella bursa), behena sativa (Eutrema salsingeeum), Brassica napus (Brassica napus), Arabidopsis thaliana (Arabis alpina), Brassica rapa (Brassica rapa), and Thladianthus urinaria (Tarenayassoseriana) having homology of more than 85% with AtGPAT1 and AtGPAT5 protein sequences, respectively.
Preferably, the construction process of the intermediate lethal yeast double mutant in the screening and identifying method of the GPATs genes comprises the following steps:
firstly, carrying out double enzyme digestion on YEplac181-GAT1-LEU2 vector by BamHI and XhoI, removing a yeast GAT1 gene, and then cloning a gene with GPAT activity in an Arabidopsis thaliana GPATs gene family to the vector through BamHI and XhoI enzyme digestion sites;
secondly, the constructed expression vectors are respectively transformed into yeast double mutants NIU8 and LEI5 to obtain the intermediate yeast double mutants containing the two expression vectors.
Preferably, the genes with GPAT activity in the Arabidopsis thaliana GPATs gene family in the screening and identifying method of the GPATs genes comprise AtGPAT1, AtGPAT5 and AtGPAT 7.
Preferably, the genetic type of the yeast double mutant NIU8 in the screening and identifying method of the GPATs gene is BY4742GAT1 Δ GAT2 Δ + [ pGAL1:: GAT1URA3, and the genetic type of LEI5 is BY4742GAT1 Δ GAT2 Δ + [ pGAL1:: GAT2URA3, and the double mutants NIU8 and LEI5 are constructed under the genetic background of the BY4742 strain.
Preferably, the intermediate yeast double mutant in the screening and identifying method of GPATs gene comprises
BY4742 gat1△gat2△+pGAL::GAT1 URA3+pGAL1::AtGPAT1 LEU2、
BY4742 gat1△gat2△+pGAL::GAT1 URA3+pGAL1::AtGPAT5 LEU2、
BY4742 gat1△gat2△+pGAL::GAT1 URA3+pGAL1::AtGPAT7 LEU2、
BY4742 gat1△gat2△+pGAL::GAT2 URA3+pGAL1::AtGPAT1 LEU2、
BY4742 gat1△gat2△+pGAL::GAT2 URA3+pGAL1::AtGPAT5 LEU2、
BY4742 gat1△gat2△+pGAL::GAT2 URA3+pGAL1::AtGPAT7 LEU2、
Preferably, the pYES2-yADH1-Kan novel plasmid in the third step of the screening and identifying method of the GPATs genes comprises the following characteristics: (1) inducing the expression of the exogenous candidate gene by taking yeast alcohol dehydrogenase 1 as a promoter (yADH1), wherein the promoter is a glucose-induced constitutive expression promoter; (2) introducing a kanamycin resistance gene as a selection marker of the shuttle plasmid in bacteria; (3) restriction sites HindIII, KpnI, SacI, BamHI, SpeI, EcoRI, NofI, XhoI, XbaI were introduced for gene cloning.
The screening and identification method of the GPATs gene is used for screening or identifying a triphosglycerol acyltransferase gene.
The screening and identification method of the GPATs gene is used for screening or identifying inhibitors of the triphosgene acyltransferase gene.
The screening and identification method of the GPATs gene is used for optimizing or modifying the sequence of the triphosglycerol acyltransferase gene.
The screening and identification method of GPATs gene is used for screening or identifying the gene participating in the first step acylation reaction enzyme in the glyceride synthesis pathway.
The more specific technical solution of the invention is as follows:
1. construction of novel conditional lethal yeast double mutants
1.1 Using the characteristic of low enzymatic activity generated BY the expression of heterologous acyltransferase (GPATs) genes in yeast to replace the genes of GPATs in yeast double mutants, construct Arabidopsis thaliana acyltransferase gene expression vectors YEplac181-AtGPAT1(pGAL1:: AtGPAT1LEU2) and YEplac181-AtGPAT5(pGAL1:: AtGPAT5LEU2) to respectively transform yeast double mutants BY-NIU8(BY4742 GAT1 △ GAT2 △ + pGAL:: GAT1URA 3) and BY-LEI5(BY4742GAT1 △ GAT2 △ + pGAL:: GAT2URA 3) to obtain intermediate yeast double mutants containing two expression vectors (such as BY4742GAT 6342 GAT2 △ + pGT 2 △: GAT 686 9A 6862 URAT 848653).
1.2 intermediate type yeast double mutants were shaken overnight in liquid medium containing Uracil (URA), partially derived from NIU8 mutant cell loss pYES2-GAT1(pGAL1:: GAT1URA 3) or derived from LEI5 mutant cell loss pYES2-GAT2(pGAL1:: GAT2URA 3), and then screened in solid medium containing 5-FOA and URA to obtain new conditional lethal yeast double mutants ZAFU1(BY4742, GAT1 △ GAT2 △ + [ pGAL1:: AtGPAT1LEU2]) and FU2(BY4742, GAT1 △ GAT2 △ + [ pGAL1:: AtGPAT5LEU2 ]).
BY4742 Yeast genotype, GAT1 and GAT2 located on yeast K and B chromosomes, respectively, GAT1 △ GAT2 △ + pGAL1, GAT1URA3 represents a yeast double mutant containing pYES2-GAT1 plasmid, GAT1 △ GAT2 △ + [ pGAL1:: AtGPAT1LEU2 represents a novel yeast double mutant containing YEplac181-AtGPAT1 plasmid.
2. Establishment of complementary system based on novel conditional lethal yeast double mutants
2.1 the novel conditional lethal yeast double mutant is not sufficient to restore growth in a medium containing galactose due to the high expression efficiency of the heterologous Arabidopsis genes (AtGPAT1 and AtGPAT5) of the novel conditional lethal yeast double mutant ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]) (GAL1 is a galactose-inducible promoter), which results in the restoration of growth of the novel conditional lethal yeast double mutant in a medium containing galactose, but is not sufficient to restore growth in a medium containing glucose (GAL1 is a glucose-repressible promoter) due to the low expression activity of the heterologous Arabidopsis genes, which is expressed as double-mutant.
2.2 based on the screening principle, the modified pYES2-yADH1-Kan novel plasmid (yADH1 is a glucose-induced expression promoter) is used for constructing a candidate GPATs gene expression vector, a novel conditional lethal yeast double mutant ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ AL1: AtGPAT5LEU2]) are transformed, and a culture medium containing glucose is used as the screening condition, so that the function of the candidate GPATs gene can be rapidly and efficiently identified.
Compared with the prior art, the method has the advantages and effects that the steps of an in vitro enzymology analysis method of the GPATs are complex, the method depends on radioactive labeling, and the screening efficiency is low, the screening system of the novel conditional lethal yeast double mutant ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]) can avoid the problems and can solve the false positive problem of the previously constructed yeast double mutant screening system, and meanwhile, the novel conditional lethal yeast double mutant is a high-specificity and high-efficiency in vivo genetic complementation screening system and has stable screening and reliable identification effects on heterologous GPATs genes.
The concrete points are as follows:
1. the conditional lethal yeast double mutant is constructed by using genes which can generate enough GPAT activity under the condition of heterologous high expression in yeast cells to restore the growth of the lethal double mutant, but cannot maintain the growth of the cells under the condition of low expression, and the conditional lethal double mutant is used for screening and identifying GPATs genes.
2. The Arabidopsis genes AtGPAT1 and AtGPAT5 are used for constructing a conditional lethal yeast double mutant, and the conditional lethal double mutant is used for screening and identifying the GPATs genes.
3. Constructing a conditional lethal yeast double mutant by using a heterologous gene with homology of more than 85 percent with AtGPAT1 and AtGPAT5, and screening and identifying GPATs genes by using the conditional lethal double mutant.
4. The GPATs gene in other non-plant heterologous species, such as mammal GPATs gene, is used for constructing a conditional lethal yeast double mutant, and the conditional lethal double mutant is used for screening and identifying the GPATs gene.
5. Novel conditional lethal yeast double mutants ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]) were used for screening and identification of GPATs.
6. Novel conditional lethal yeast double mutants ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]) were used for the identification of the key sequences for the enzymatic activity of GPATs.
7. Novel conditional lethal yeast double mutants ZAFU1(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT1LEU2]) and ZAFU2(BY4742, gat1 △ gat2 △ + [ pGAL1:: AtGPAT5LEU2]) are applied to screening and identification of GPATs inhibitors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of construction of the novel yeast double mutant of the present invention.
FIG. 2 shows the restriction enzyme digestion verification electrophoretograms of the vectors PCR2.1-GAT1 and PCR2.1-GAT1 of the present invention.
FIG. 3: restriction enzyme electrophorograms of expression vectors pYES2-GAT1 and pYES2-GAT 2.
FIG. 4: the drawing of the map of the expression vector pYES2-GAT1 shows.
FIG. 5: GAT1 and GAT2 gene knockout vectors are schematically constructed.
FIG. 6: enzyme cutting electrophoresis picture of GAT1 and GAT2 gene knockout carrier
FIG. 7: enzyme cutting electrophoresis picture of expression vector YEplac181-GAT1
FIG. 8: YEplac181-GAT1 vector map
FIG. 9: lei5, lei7 mutant PCR verified primer position map
FIG. 10: PCR-verified PCR electrophoretogram of lei5 and lei7 mutants
FIG. 11: niu7, niu8 mutant PCR verified primer position map
FIG. 12: niu7, niu8 mutant PCR verification electrophoretogram
FIG. 13: growth state diagram of double mutants and single mutant on 5-FOA medium
FIG. 14: pYES2-Kan-yADH1 expression vector map
FIG. 15: screening efficiency map of novel conditional lethal yeast double-mutant genetic complementation system
FIG. 16A: linear graph of influence of deletion of N-terminal amino acid residue on AtGAPT1 enzyme activity
FIG. 16B: effect of deletion of N-terminal amino acid residue on AtGAPT1 enzymatic Activity
Brief description of the drawingsthe accompanying drawings:
FIG. 1 shows BY4742 yeast genotype, GAT1 and GAT2 located on yeast K and B chromosomes, respectively, GAT1 △ GAT2 △ + pGAL1, GAT1URA3 represents a yeast double mutant containing pYES2-GAT1 plasmid, GAT1 △ GAT2 △ + [ pGAL1:: AtGPAT1LEU2 represents a novel yeast double mutant containing YEplac181-AtGPAT1 plasmid.
FIG. 2: the genes GAT1 and GAT2 were amplified using yeast genome as template and ligated to PCR2.1 vector by T-cloning.
1: PCR2.1-GAT1(BamHI and XhoI);
2: PCR2.1-GAT1(BamHI and XhoI);
M：DNA Marker
FIG. 3: the GAT1 and GAT2 fragments were excised from the vectors PCR2.1-GAT1 and PCR2.1-GAT1 with BamHI + XhoI and ligated to the expression vector pYES2, respectively.
1、2、3、4：pYES2-GAT1(BamHI+XhoI)；
5、6、7、8：pYES2-GAT2(BamHI+XhoI)；
M：DNA Marker
FIG. 4: genetic map of expression vector pYES2-GAT 1.
FIG. 5: and (2) amplifying to obtain an HIS3 fragment by taking pRS423 as a template, amplifying to obtain a 5 '-GAT 1-PCR2.1-GAT 1-3' fragment by taking PCR2.1-GAT1 as a template, carrying out double enzyme digestion and connection by SmaI and PstI to obtain a gene knockout vector PCR2.1-5 '-GAT 1-PCR2.1-GAT 1-3', and carrying out enzyme digestion electrophoresis verification. The same way is adopted to obtain the knockout vector PCR2.1-5 '-GAT 2-PCR2.1-GAT 2-3'.
FIG. 6: enzyme cutting electrophoresis picture of GAT1 and GAT2 gene knockout carrier
1: GAT1 gene knock-out vector (SmaI + PstI);
2: GAT2 gene knock-out vector (SamI + PstI);
M：DNA Marker
FIG. 7: amplifying by taking pYES2-GAT1 as a template to obtain pGAL1-GAT1-CYCTT fragments, carrying out double enzyme digestion on the pGAL1-GAT1-CYCTT fragments and a YEplac181 vector by PstI and SmaI, connecting to obtain an expression vector YEplac181-GAT1, and carrying out double enzyme digestion verification by XhoI and BamHI;
M：DNA Marker；1：YEplac181-GAT1(XhoI+BamHI)
FIG. 8: YEplac181-GAT1 vector map
FIG. 9: lei5 and lei7 mutants were verified by PCR, and the mutant genomes were extracted and verified by three pairs of primers, respectively, which are position maps of the verification primers.
FIG. 10: reprinted PCR electrophoretogram for PCR verification of ei5, lei7 mutants
1: lei5 genome as template
2: lei7 genome as template
1. 2: ZZF27+ ZZF10 as primers
1 ', 2': ZZF28+ ZZF9 as primers
1 ', 2': ZZF27+ ZZF28 as primers
M1：2.0bp DNA Marker
M2：10kb DNA Marker
FIG. 11: niu7 and niu8 mutants were verified by PCR, and the mutant genomes were extracted and verified by three pairs of primers, respectively, which are position maps of the verification primers.
FIG. 12: niu7, niu8 mutants,
1: niu7 genome as template
2: niu8 genome as template
1. 2: ZZF29+ ZZF9 as primers
1 ', 2': ZZF30+ ZZF10 as primers
1 ', 2': ZZF29+ ZZF30 as primers
M1：2.0bp DNA Marker
M2：10kb DNA Marker
FIG. 13: the growth state diagram of the double mutant and the single mutant on the 5-FOA culture medium is that the OD600 is 0.5 bacterial liquid, and the bacterial liquid is diluted by4 times and 5 concentration gradients are respectively spotted on different SC culture media for 22 or 33 hours.
FIG. 14: pYES2-Kan-yADH1 expression vector map
FIG. 15: screening efficiency of novel conditional lethal yeast double-mutant genetic complementation system
FIG. 16 includes A and B: effect of deletion of N-terminal amino acid residues on AtGAPT1 enzymatic activity. (A) Growth curve analysis of yeast double mutants with the wild type gene and mutant gene of AtGAPT1 (5' end 1-237 base deletion); (B) and (3) analyzing growth difference of the cells with 4 genetic types under different cell concentrations, wherein the 4 genetic types are yeast double mutants with blank plasmids, yeast double mutants with wild type AtGAPT1 genes, yeast mutants with 5 'end 1-237 base deletion AtGAPT1 genes and 5' end 1-372 base deletion AtGAPT1 genes. Initial cell concentration OD600Dilution was 1:5, 0.5.
Detailed Description
The present invention will be described in further detail with reference to examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
Example (b):
1. construction of a genetic complementation System
1.1 expression vector construction
1.1.1 construction of expression vectors pYES2-GAT1 and pYES2-GAT2
The vector PCR2.1-GAT1 and PCR2.1-GAT1 are formed by amplifying a GAT1 fragment and a GAT2 FP and GAT2RP fragment by using a yeast genome as a template and GAT1 FP and GAT1 RP as primers and respectively connecting the amplified GAT2 fragments to a vector PCR2.1 by TA cloning, and verified by BamHI and XhoI double digestion, and the electrophoresis is shown in figure 2, wherein the size of the GAT1 fragment is 2.2kb, the size of the GAT2 fragment is about 2.2kb, and the size of the PCR2.1 is about 3.9 kb.
The PCR2.1-GAT1 and pYES2 vectors were double digested with BamHI and XhoI restriction enzymes, two fragments of GAT1 and pYES2 were recovered, ligated to give the expression vector pYES2-GAT1, and verified by double digestion with BamHI and XhoI, and electrophoresed as shown in FIG. 3, GAT1 about 2.2kb and pYES2 about 5.9 kb.
The PCR2.1-GAT2 and pYES2 vectors were double digested with BamHI and XhoI restriction enzymes, two fragments of GAT2 and pYES2 were recovered, ligated to give the expression vector pYES2-GAT2, and verified by double digestion with BamHI and XhoI, and electrophoresed as shown in FIG. 3, GAT2 about 2.2kb and pYES2 about 5.9 kb.
1.1.2 construction of GAT1 and GAT2 Gene knockout vectors
Amplifying a 5 '-GAT 1-PCR2.1-GAT 1-3' fragment by taking PCR2.1-GAT1 as a template and ZZF11 and ZZF12 as primers; the HIS3 fragment was amplified by ZZF15 and ZZF16 using pRS423 as a template, and simultaneously double-digested with SamI and PstI, and then ligated together to construct a new vector PCR2.1-5 '-GAT 1-HIS3-GAT 1-3', and verified by double-digestion with SamI and PstI, as shown in FIG. 6, the size of 5 '-GAT 1-HIS3-GAT 1-3' fragment was about 2.0kb, and the size of PCR2.1 was about 3.9 kb. Similarly, using PCR2.1-GAT2 as a template and ZZF13 and ZZF14 as primers, amplifying a 5 '-GAT 2-PCR2.1-GAT 2-3' fragment, simultaneously double-digesting with SamI and PstI, and then connecting to form a new vector PCR2.1-5 '-GAT 2-HIS3-GAT 2-3', and verifying by double-digesting with SamI and PstI, wherein the electrophoresis is as shown in FIG. 6, the size of the 5 '-GAT 2-HIS3-GAT 2-3' fragment is about 2.0kb, and the size of the PCR2.1 is about 3.9 kb.
PCR2.1-5 ' -GAT1-HIS3-GAT1-3 ' is used as a template, ZZF17 and ZZF18 are used as primers, 5 ' -GAT1-HIS3-GAT1-3 fragments are obtained by high-fidelity enzyme amplification, and the fragments are recovered for standby use and are used for knocking out the GAT1 gene in a yeast genome.
PCR2.1-5 '-GAT 2-HIS3-GAT 2-3' is used as a template, ZZF19 and ZZF20 are used as primers, 5 '-GAT 2-HIS3-GAT 2-3' fragments are obtained by high-fidelity enzyme amplification, and the fragments are recovered for standby use and are used for knocking out the GAT2 gene in a yeast genome.
1.1.3 construction of expression vector YEPlac181-GAT1
Using pYES2-GAT1 as template and ZZF45 and ZZF46 as primers, GAL1-GAT1-CYC1TT (promoter + GAT1 fragment + terminator) fragment was obtained by high fidelity enzymatic amplification. The GAL1-GAT1-CYC1TT fragment and YEplac181 were digested simultaneously with PstI and SmaI to obtain an overexpression vector YEplac181-GAT1, which was verified by BamHI + XhoI digestion, and electrophoresed as shown in FIG. 7.
1.2. Construction of conditional double mutants
The vector pYES2-GAT2 is transferred into a GAT2 △ mutant, and a new mutant GAT2 △ + [ pGAL1:: GAT2URA3] is obtained by screening an SC-URA + Gal (galactose) culture medium, a GAT1U-HIS3-GAT1D fragment is introduced into a mutant GAT1 △ GAT2 △ + [ pGAL1:: GAT2URA3] by using a homologous recombination principle, a GAT1 gene in the mutant is replaced, screening is carried out by an SC-URA (uracil) -His (histidine) + Gal culture medium, and PCR verification is carried out by using primers 1+ 1, 1+ 1 and 1+ 1 (figure 9), and electrophoresis is carried out as shown in figure 10, so as to obtain two positive clones GAT 1GAT 1+ [ pGAL1:: GAT2URA 1:, LEI 1-1.
The vector pYES2-GAT1 was transferred into the GAT1 △ mutant, and the new mutant GAT1 △ + [ pGAL1:: GAT1URA 3] was obtained by screening with SC-URA + Gal medium, using the principle of homologous recombination, GAT2U-His-GAT2D fragment was introduced into mutant GAT1 △ GAT2 △ + [ pGAL1:: GAT1URA 3], replacing the GAT2 gene in the mutant, screening with SC-URA-His + Gal medium, and PCR verification was performed with primers ZZF29+ ZZF9, ZZF30+ ZZF10 and ZZF29+ ZZF30, and electrophoresis is shown in FIG. 12, thus obtaining two positive clones GAT1 △ GAT2 △ + [ pGAL1:: GAT1URA 3], named as 39 niu8-1, niu 8-2.
1.3 FOA validation of double mutants
5-FOA as a negative screening drug, when yeast cells can express URA3, URA3 gene encodes important enzyme in uracil synthesis pathway: orotidine 5-phosphate decarboxylase, which converts 5-fluoroorotate (5-FOA) into a cytotoxic substance, rendering the cell incapable of growing. Conversely, the cells can grow.
When the strain is shaken by using the URA-containing SC medium, part of the pYES2 plasmids in GAT1 △ + pYES2, GAT2 △ + pYES2-URA3 and GAT1 △ + pYES1-GAT2 are lost to form GAT1 △, GAT2 △ and GAT1 △ mutants respectively, and the mutants can grow on the URA-containing medium, while the yeasts without losing the pYES2 plasmids can express URA3, cannot grow on the 5-FOA-containing medium and can grow on the 5-FOA-free medium.
The strain was shaken with SC medium containing URA to lose part of pYES2 plasmid in LEI5 and NIU8 and thus prevent growth, while those without losing pYES2 plasmid expressed URA3, which did not grow on medium containing 5-FOA and did not grow on medium containing 5-FOA.
From the above results, we can derive: in the above 5-FOA screening system, the survival of the double mutant is dependent on the expression of GAT1 or GAT2 gene. The combined expression vector of the target gene and YEplac181 is transferred into LEI5, the SC culture medium containing URA is firstly used for shaking bacteria, the pYES2 plasmid is lost, and the mutant obtained by 5-FOA screening is introduced, wherein the target gene is a gene with similar functions to GAT1 or GAT 2. Therefore, a new GPATs screening system is established, and the target gene is screened in the next step.
2. Construction of novel conditional lethal yeast double mutants
The YEplac181-GAT1-LEU2 vector (FIG. 8) was double-digested with BamHI and XhoI, the yeast GAT1 gene was deleted, and then the genes having GPAT activity in the Arabidopsis GPATs gene family (AtGPAT1, AtGPAT4, AtGPAT5, AtGPAT6, AtGPAT7, AtGPAT8) were cloned into the vector through both BamHI and XhoI cleavage sites. The newly constructed expression vector has the following characteristics: i the foreign gene is driven by a galactose (GAL1) inducible promoter; ii the corresponding CYC terminator is introduced at the same time.
The constructed expression vectors were transformed into the yeast double mutants NIU8(BY4742 GAT1 Δ GAT2 Δ + [ pGAL1:: GAT1URA 1 ]) and LEI 1(BY4742 GAT1 Δ GAT1 Δ + [ pGAL1:: GAT2URA 1:: 1), the intermediate yeast double mutants GAT 1:: GAT 473672 GAT 1+ GAGAT 1+ 1 pGGAGAGAGAT 1:: GAGAGAT 36473672:: pGGAGAGAGAT 36473672 + pGGAGAT 363636363672:: GAGAGAGAGAGAGAT 1+ 368 GPAT1, the GAGAGAGAGAGAT 36473672:: pG364736363672 + pGGAGAGAGAGAGAGAGAGAGAGAGAT 1:, the intermediate yeast double mutants GAT 3636363672 pG3636363636363636363636363636363636363672:, the pGGABYt 3636363636363636363636363636363636363636363636368 + GAGABYGAGAGAGAGAGAGAGAGAGAGAGAYG3672:, the No 36363636363636363636363636363636363636363672 plus 3636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636368 and the No 36363636363636363636363636363636368 and the No 368 pGGAYGweight, the No 363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636363636368, the No 36363636368, the yeast GAYGweight, the yeast.
The intermediate yeast double mutant is shaken overnight in SC liquid culture which contains galactose and uracil and does not contain histidine and leucine, about 1 percent of the intermediate yeast double mutant loses expression vector pGAL containing uracil screening marker, GAT1URA3 or pGAL, GAT2URA 3; then, the cells were plated on galactose solid medium plates containing uracil and 5-fluoroorotic acid and not containing histidine and leucine and glucose solid medium plates containing uracil and 5-fluoroorotic acid and not containing histidine and leucine, respectively. And the yeast cells without losing the uracil selection marker expression vector can not grow in the culture medium containing 5-fluoroorotic acid because 5-fluoroorotic acid is converted into toxic substances by the expression of uracil gene. The above screening results showed that some of the yeast cells derived from the primary genotype double mutants transformed with the vectors YEPlac181-AtGPAT1, YEPlac181-AtGPAT5 and YEPlac181-AtGPAT7 grew normally in a galactose-containing medium, but did not grow at all in a glucose-containing medium; and the growth speed of the primitive genotype double mutant derived from the YEPlac181-AtGPAT7 transformation vector is obviously slower than that of the primitive genotype double mutant derived from the YEPlac181-AtGPAT1 and YEPlac181-AtGPAT5 transformation vectors; in addition, the primary genotype double mutants derived from the transformation vectors YEPlac181-AtGPAT4, YEPlac181-AtGPAT6 and YEPlac181-AtGPAT8 did not grow in both galactose and glucose media (Table 1).
PCR detection of the URA3, GAT1, AtGPAT1, AtGPAT5, and AtGPAT7 genes was performed on randomly picked single clones of primary genotype double mutants derived from vectors transformed with YEPlac181-AtGPAT1, YEPlac181-AtGPAT5, and YEPlac181-AtGPAT 7. The results showed that yeast monoclonals derived from the transformants YEPlac181-AtGPAT1, YEPlac181-AtGPAT5, and YEPlac181-AtGPAT7 contained Arabidopsis thaliana foreign genes AtGPAT1, AtGPAT5, and AtGPAT7, respectively; among them, the original genetic double mutant derived from the vectors YEPlac181-AtGPAT1 and YEPlac181-AtGPAT5, which contained no URA3 and GAT1 genes, and only 1 of 5 single clones derived from the vectors YEPlac181-AtGPAT7, which contained GAT1 genes (Table 1), showed that the Arabidopsis AtGPAT1, AtGPAT5 and AtGPAT7 genes had the same GPAT catalytic activity as that of yeast GAT1 and GAT2, resulting in the newly formed yeast double mutants (GAT1 Δ GAT2 Δ + [ pGAL1:: AtGPAT1LEU2], GAT1 Δ GAT 2+ [ pGAL1:: AtGPAT5LEU2] and GAT1 Δ GAT 2+ [ pGAL1: [ AtGPAT 7U 2: ] which induced lower growth of glucose in a galactose medium and thus did not recover the exogenous glucose growth activity.
We named the newly formed conditional lethal yeast double mutant gat1 Δ gat2 Δ + [ pGAL1:: AtGPAT1LEU2, gat1 Δ gat2 Δ + [ pGAL1:: AtGPAT5LEU2 and gat1 Δ gat2 Δ + [ pGAL1:: AtGPAT7LEU2 ZAFU1, ZAFU2 and ZAFU3, respectively.
TABLE 1 phenotype of NIU8 hereditary form of yeast double mutant with self GAT1 gene replaced by Arabidopsis AtGPAT Gene family and PCR detection results
Source ofPrimitive genetical double mutant Yeast cells transformed with YEPlac181-AtGPAT1 and YEPlac181-AtGPAT5 plated at 1X10 on SC + URA-His-leu + gal + FOA solid Medium6Individual cells, control and other candidate genes plated at 5X107And (4) cells.
The ratio of the cells containing the target gene to the total cells to be detected
3. Establishment of complementary system of novel conditional lethal yeast double mutants
Based on a novel conditional lethal yeast double mutant ZAFU1(gat1 Δ gat2 Δ + [ pGAL1:: AtGPAT1LEU2), a yeast in vivo genetic complementation system for screening heterologous GPATs genes is constructed by utilizing a glucose specific induction promoter expression vector. We have successfully engineered and obtained the expression vector pYES2-Kan-yADH1 (FIG. 14) for the genetic complementation screen of conditional lethal yeast double mutants, which has the following characteristics: i, inducing the expression of the exogenous candidate gene by taking yeast alcohol dehydrogenase 1 as a promoter (yADH1), wherein the promoter is a glucose-induced constitutive expression promoter; ii, kanamycin is introduced into the expression vector to serve as a prokaryotic expression screening marker, and the ampicillin screening marker is different from the ampicillin screening marker of the complementary vector of the conditional lethal yeast double mutant and is easy to separate candidate complementary genes; and iii, introducing multiple cloning sites HindIII, KpnI, SacI, BamHI, SpeI, EcoRI, NofI, XhoI and XbaI, and facilitating cloning of exogenous selection genes.
We cloned 4 known yeast genes (GAT1, GAT2, SCL1 and PST1) and an Arabidopsis thaliana gene (LPAT4) into pYES2-Kan-yADH1 expression vector and transformed into a novel conditional lethal yeast double mutant ZAFU1 using glucose-containing medium as screening conditions. The results show that transformation of the yeast GAT1 and GAT2 genes with GAPT catalytic activity restored growth of the conditional lethal yeast double mutant ZAFU1 in glucose medium, while the remaining genes without GAPT catalytic activity did not restore growth of the conditional lethal yeast double mutant ZAFU1 (Table 2). In addition, the conditional lethal yeast double mutant ZAFU1 of the transformed yeast GAT1 and GAT2 genes can recover the growth in glucose culture, the growth rate of the mutant in galactose culture medium is improved, the growth rate of the mutant is closely related to the GAPT catalytic activity, and meanwhile, the novel conditional lethal yeast double mutant constructed by the method is a very feasible, stable and specific eukaryotic GAPTs gene screening system.
TABLE 2 GPAT Gene Screen based on a novel conditional lethal Yeast double mutant genetic complementation screening System
We further tested the high efficiency of the new conditional lethal yeast double mutant ZAFU1 genetic complementation screening system using yeast GAT1 gene. An empty vector (control), a pYES2-Kan-yADH1+ GAT1 expression vector and a vector mixed by the two vectors according to the proportion of 1:1000 and 1:5000 are transformed into a novel conditional lethal yeast double mutant ZAFU1, and then the culture medium screening of galactose and glucose is carried out. The results show that the expression vector condition lethal yeast double mutant ZAFU1 transformed with pYES2-Kan-yADH1+ GAT1 can well recover the growth, while the control has no cell growth at all (FIG. 15); meanwhile, the empty vector (control) and the pYES2-Kan-yADH1+ GAT1 expression vector can also obtain a better positive complementation test result under the condition that the concentration is 1:1000 (figure 15), which shows that the novel conditional lethal yeast double-mutant ZAFU1 genetic complementation screening system has higher screening efficiency.
4. Discovery of key amino acid residues controlling AtGPAT1 enzymatic activity
We clone the mutant with deletion of different length base pairs at the 5 'end of the AtGPAT1 gene into a pYES2-Kan-yADH1 vector, then transform a conditional lethal mutant ZAFU1, and as a result, the result shows that the deletion of 237 base pairs at the 5' end of the AtGPAT1 gene (corresponding to amino acid residues 1-79 at the N end of the polypeptide) can restore the growth of the yeast mutant, and the AtGPAT1 mutant is the same as the wild AtGPAT1, thus showing that the amino acid residues 1-79 at the N end do not obviously influence the activity of the GPAT enzyme (FIGS. 16A and B). However, when the 372 base pairs at the 5' end of AtGPAT1 gene (corresponding to amino acid residues 1-124 at the N-terminus of the polypeptide) were deleted, the mutant AtGPAT1 gene failed to restore the growth ability of the conditionally lethal yeast mutant, thus indicating that amino acid residues 80-124 at the N-terminus of the polypeptide are essential for GPAT enzyme activity.
Furthermore, it should be noted that the embodiments described in the present specification, and the names and the like of some substances mentioned therein may be different, and all the equivalent or simple changes made according to the features and the principle described in the present patent idea are included in the protection scope of the present patent, and those skilled in the art of the present invention may make various modifications or additions or substitute the similar way for the described embodiments without departing from the structure of the present invention or exceeding the scope defined by the claims.
Claims (13)
1. A method for screening and identifying a 3-phosphoglycerate acyltransferase (GPATs) gene, comprising the steps of:
step one, constructing an intermediate yeast double mutant: introducing an expression vector with a heterologous GPATs gene into a yeast double mutant of an expression vector with a yeast self GAT1 or GAT2 gene by a yeast genetic transformation means to obtain an intermediate yeast double mutant containing two expression vectors with a yeast GAT1 or GAT2 gene and a heterologous GPAT gene respectively; the yeast double mutant of the expression vector with the yeast self GAT1 or GAT2 gene refers to a yeast double mutant which is generated by knocking out the GAT1 and GAT2 genes on the yeast self genome and has the expression vector with the yeast self GAT1 or GAT2 gene;
step two, shaking the intermediate yeast double mutant obtained in the step one through a uracil-Urea (URA) -containing liquid culture medium overnight, and then screening on a solid culture medium containing 5-fluoroorotic acid (5-FOA) and URA to obtain a new conditional lethal yeast double mutant of the expression vector containing a heterologous GPAT gene;
thirdly, constructing a candidate GPATs gene expression vector by using the modified pYES2-yADH1-Kan novel plasmid, and introducing the candidate GPATs gene expression vector into a new conditional lethal yeast double mutant;
and step four, adopting a culture medium containing glucose as a screening condition, and enabling the new conditional lethal yeast double mutant to recover the growth of a heterologous gene in the glucose culture medium, namely the gene encoding the GPAT enzyme activity.
2. The method for screening and identifying GPATs gene as claimed in claim 1, wherein the heterologous GPATs gene in step one is AtGPAT1 and AtGPAT5 in Arabidopsis thaliana.
3. The method for screening and identifying GPATs gene as claimed in claim 1, wherein the homology between the protein sequence encoded by the heterologous GPATs gene in step one and the protein sequences of AtGPAT1 and AtGPAT5 in Arabidopsis thaliana is up to 85% or more.
4. The method for screening and identifying GPATs gene according to claim 3, wherein said gene having more than 85% homology with AtGPAT1 and AtGPAT5 protein sequence in Arabidopsis thaliana comprises the gene sequence having more than 85% homology with AtGPAT1 and AtGPAT5 protein sequence in Arabidopsis thaliana (Arabidopsis thaliana), Camelina sativa (Camelina sativa), Camelina sativa (Capsella bursa), behena (Eutreemaselguinum), Brassica napus (Brassica napus), Arabidopsis thaliana (Arabis alpina), Brassica napus (Brassica), and Thauba (Tarrenaya hassleyana), respectively.
5. The method for screening and identifying the GPATs gene as claimed in claim 1, wherein the construction process of the intermediate lethal yeast double mutant comprises:
5.1, carrying out double enzyme digestion on YEplac181-GAT1-LEU2 vector by BamHI and XhoI, removing a yeast GAT1 gene, and cloning a gene with GPAT activity in an Arabidopsis thaliana GPATs gene family to the vector through BamHI and XhoI enzyme digestion sites to obtain an expression vector;
5.2, respectively transforming the expression vectors obtained in the step 5.1 into yeast double mutants NIU8 and LEI5 to obtain intermediate yeast double mutants containing the two expression vectors.
6. The screening and identification method of GPATs gene as claimed in claim 5, wherein the genes having GPAT activity in Arabidopsis thaliana GPATs gene family include AtGPAT1, AtGPAT5, AtGPAT 7.
7. The screening and identification method of GPATs gene as claimed in claim 5, characterized in that the genetic type of the yeast double mutant NIU8 is BY4742GAT1 Δ GAT2 Δ + [ pGAL1:: GAT1URA3, the genetic type of LEI5 is BY4742GAT1 Δ GAT2 Δ + [ pGAL1:: GAT2URA3, and the double mutants NIU8 and LEI5 are constructed under the genetic background of BY4742 strain.
8. The method for screening and identifying the GPATs gene as claimed in claim 5, wherein the intermediate yeast double mutant comprises
BY4742gat1△gat2△+pGAL::GAT1URA3+pGAL1::AtGPAT1LEU2、
BY4742gat1△gat2△+pGAL::GAT1URA3+pGAL1::AtGPAT5LEU2、
BY4742gat1△gat2△+pGAL::GAT1URA3+pGAL1::AtGPAT7LEU2、
BY4742gat1△gat2△+pGAL::GAT2URA3+pGAL1::AtGPAT1LEU2、
BY4742gat1△gat2△+pGAL::GAT2URA3+pGAL1::AtGPAT5LEU2、
BY4742gat1△gat2△+pGAL::GAT2URA3+pGAL1::AtGPAT7LEU2。
9. The method for screening and identifying the GPATs gene as claimed in claim 1, wherein the pYES2-yADH1-Kan novel plasmid in step three comprises the following characteristics: (1) inducing the expression of the exogenous candidate gene by taking yeast alcohol dehydrogenase 1 as a promoter, wherein the promoter is a glucose-induced constitutive expression promoter; (2) introducing a kanamycin resistance gene as a selection marker of the shuttle plasmid in bacteria; (3) restriction sites HindIII, KpnI, SacI, BamHI, SpeI, EcoRI, NofI, XhoI, XbaI were introduced for gene cloning.
10. A method for screening or identifying a glycerol triphosphate acyltransferase gene using the GPATs gene of any one of claims 1 to 9.
11. A method for screening or identifying the inhibitors of the glycerol triphosphate acyltransferase gene using the GPATs gene of any one of claims 1 to 9.
12. A method for screening and identifying the GPATs gene as claimed in any one of claims 1 to 9 for optimizing or modifying the sequence of the triphosglycerol acyltransferase gene.
13. A method for screening or identifying a gene involved in the first step of an acylation reaction enzyme in a glycerolipid synthesis pathway, which comprises using the GPATs gene screening and identifying method according to any one of claims 1 to 9.
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