酿酒酵母起始密码子表达载体pYES2-ATG的构建及应用
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摘要
鉴定合成的或通过高通量随机组合方法获得的潜在功能片段的生物学功能需要一套高效便捷的酵母表达专用载体。酵母表达载体pYES2多克隆位点处不含起始密码子,通过设计含有ATG和Eco RⅠ酶切位点的特异性引物扩增一段Intron,利用In-Fusion重组技术(Clontech)将该片段构建至酵母表达载体p YES2,得到一个含有ATG的重组酵母表达载体pYES2-ATG。为了检测该载体的功能,扩增不含起始密码子的DNA序列nlea,该序列由本实验室设计合成,具有潜在的耐盐功能。构建重组表达载体p YES2-ATG-nlea在酵母中进行功能鉴定。研究结果表明,半乳糖诱导后,含pYES2-ATG-nlea的重组酵母菌株耐盐能力明显高于含pYES2-ATG空质粒的菌株,表明在该载体系统上的添加了起始密码子的nlea成功表达,具有一定的耐盐性,同时也证明改造的载体系统可用于没有起始密码子的编码区序列的功能筛选和鉴定。p YES2-ATG酵母载体系统不影响原始载体基本功能元件的表达,同时能使不含起始密码子编码区序列在酵母中正常表达,在大规模进行多个基因的功能鉴定中具有重要的应用价值。
Identification of the biological functions of potential functional fragments obtained by synthetic or high-throughput random combination requires a set of efficient and convenient yeast expression vectors. Yeast expression vector pYES2 polyclonal site didn't contain a start codon. Hence we obtained a recombinant yeast expression vector p YES2-ATG containing ATG by designing a specific primer containing ATG and Eco R Ⅰenzyme cutting site to amplified a fragment of nonsense intron, and then integrating it into yeast expression vector p YES2 using In-Fusion recombination technology(Clontech). In order to detect the function of the vector, the DNA sequence with no initial codon was amplified, which was designed and synthesized by our laboratory and had the potential salt-tolerant function. The recombinant expression vector pYES2-ATG-nlea was constructed for functional identification in yeast. The results showed that, after induction with galactose, the salt-tolerant ability of recombinant yeast strain containing pYES2-ATG-nlea was significantly higher than that of the strain containing pYES2-ATG empty plasmid, indicating that the nlea with initial codon was successfully expressed in the vector system and had a certain salt tolerance. Meanwhile it proved that the modified vector could be used for functional screening and identification of the coding regions sequences without initiation codon. pYES2-ATG yeast vector system didn't affect the expression of the basic functional elements of the original vector, and meanwhile, it could make the sequence of coding region without the initial codon express normally in yeast. It could have important application value in the large-scale functional identification of multiple genes.
Identification of the biological functions of potential functional fragments obtained by synthetic or high-throughput random combination requires a set of efficient and convenient yeast expression vectors. Yeast expression vector pYES2 polyclonal site didn't contain a start codon. Hence we obtained a recombinant yeast expression vector p YES2-ATG containing ATG by designing a specific primer containing ATG and Eco R Ⅰenzyme cutting site to amplified a fragment of nonsense intron, and then integrating it into yeast expression vector p YES2 using In-Fusion recombination technology(Clontech). In order to detect the function of the vector, the DNA sequence with no initial codon was amplified, which was designed and synthesized by our laboratory and had the potential salt-tolerant function. The recombinant expression vector pYES2-ATG-nlea was constructed for functional identification in yeast. The results showed that, after induction with galactose, the salt-tolerant ability of recombinant yeast strain containing pYES2-ATG-nlea was significantly higher than that of the strain containing pYES2-ATG empty plasmid, indicating that the nlea with initial codon was successfully expressed in the vector system and had a certain salt tolerance. Meanwhile it proved that the modified vector could be used for functional screening and identification of the coding regions sequences without initiation codon. pYES2-ATG yeast vector system didn't affect the expression of the basic functional elements of the original vector, and meanwhile, it could make the sequence of coding region without the initial codon express normally in yeast. It could have important application value in the large-scale functional identification of multiple genes.
引文
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Khalife S.,Aliouat el M.,Gantois N.,Jakobczyk H.,Demay F.,Chabe M.,Pottier M.,Dabboussi F.,Hamze M.,Dei-Cas E.,Standaert-Vitse A.,and Aliouat-Denis C.M.,2014,Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene,Fungal Biol.,118(11):885-895
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Miura N.,Kaneko S.,Hosoya S.,Furuchi T.,Miura K.,Kuge S.,and Naganuma A.,1999,Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae,FEBS L-ett.,458(2):215-218
Murakami N.,Miyoshi S.,Yokoyama R.,Hoshida H.,Akada R.,and Ogata T.,2012,Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus,Yeast,29(5):155-165
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Pich O.Q.,Burgos R.,Planell R.,Querol E.,and Pinol J.,2006Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium:application to studies of the minimal gene complement,Microbiology,152(2):519-527
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Yin J.,Li X.,Zhan Y.,Li Y.,Qu Z.,Sun L.,Wang S.,Yang J.,and Xiao J.,2017,Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch,BMC Plant Biology,17:214
Bauerle C.,Magembe C.,and Briskin D.P.,1998,Characterization of a red beet protein homologous to the essential 36-kilodalton subunit of the yeast V-type ATPase,Plant Physiol.,117(3):859-867
Brands A.,and Ho T.H.,2002,Function of a plant stress-induced gene,HVA22.Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic,Plant Physiol.,130(3):1121-1131
Fraser C.M.,Gocayne J.D.,White O.,Adams M.D.,Clayton R.A.,Fleischmann R.D.,Bult C.J.,Kerlavage A.R.,Sutton G.,K-elley J.M.,Fritchman R.D.,Weidman J.F.,Small K.V.,Sandusky M.,Fuhrmann J.,Nguyen D.,Utterback T.R.,Saudek D.M.,Phillips C.A.,Merrick J.M.,Tomb J.F.,Dougherty B.A.,Bott K.F.,Hu P.C.,Lucier T.S.,Peterson S.N.,Smith H.O.,Hutchison C.A.,and Venter J.C.,1995,The minimal gene complement of Mycoplasma genitalium,Science,270(5235):397-403
Gietz D.,St Jean A.,Woods R.A.,and Schiestl R.H.,1992,Improved method for high efficiency transformation of intact yeast cells,Nucleic Acids Research,20(6):1425
Hontz R.D.,Niederer R.O.,Johnson J.M.,and Smith J.S.,2009,Genetic identification of factors that modulate ribosomal DNA transcription in Saccharomyces cerevisiae,Genetics,182(1):105-119
Johnson J.M.,Smith J.S.,and Schneider D.A.,2014,A user's guide to the ribosomal DNA in Saccharomyces cerevisiae,Methods Mol.Biol.,1205:303-328
Khalife S.,Aliouat el M.,Gantois N.,Jakobczyk H.,Demay F.,Chabe M.,Pottier M.,Dabboussi F.,Hamze M.,Dei-Cas E.,Standaert-Vitse A.,and Aliouat-Denis C.M.,2014,Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene,Fungal Biol.,118(11):885-895
Li X.,Millson S.H.,Coker R.D.,and Evans I.H.,2006,Cloning and expression of Penicilliumminioluteum dextranase in Saccharomyces cerevisiae and its exploitation as a reporter in the detection of mycotoxins,Biotechnol.Lett.,28(23):1955-1964
Miura N.,Kaneko S.,Hosoya S.,Furuchi T.,Miura K.,Kuge S.,and Naganuma A.,1999,Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae,FEBS L-ett.,458(2):215-218
Murakami N.,Miyoshi S.,Yokoyama R.,Hoshida H.,Akada R.,and Ogata T.,2012,Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus,Yeast,29(5):155-165
Park J.,Throop A.L.,and LaBaer J.,2015,Site-specific recombinational cloning using gateway and in-fusion cloning schemes,Curr.Protoc.Mol.Biol.,110:1-23
Parret A.H.,Besir H.,and Meijers R.,2016,Critical reflections on synthetic gene design for recombinant protein expression,Curr.Opin.Struc.Biol.,38:155-162
Pich O.Q.,Burgos R.,Planell R.,Querol E.,and Pinol J.,2006Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium:application to studies of the minimal gene complement,Microbiology,152(2):519-527
Ray S.S.,Bandyopadhyay S.,and Pal S.K.,2012,A weighted power framework for integrating multisource information gene function prediction in yeast,IEEE Trans.Biomed Eng.,59(4):1162-1168
Sleight S.C.,Bartley B.A.,Lieviant J.A.,and Sauro H.M.,2010In-Fusion BioBrick assembly and re-engineering,Nucleic Acids Res.,38(8):2624-2636
Watanabe Y.,Iwaki T.,Shimono Y.,Ichimiya A.,Nagaoka Y.and Tamai Y.,1999,Characterization of the Na+-ATPase gene(ZENA1)from the salt-tolerant yeast Zygosaccharomyce srouxii,J.Biosci.Bioeng.,88(2):136-142
Wyrick J.J.,2012,Computational analysis of promoter elements and chromatin features in yeast,Methods Mol.Biol.,809217-235
Xie Z.X.,Li B.Z.,Mitchell L.A.,Wu Y.,Qi X.,Jin Z.,Jia B.,Wang X.,Zeng B.X.,Liu H.M.,Wu X.L.,Feng Q.,Zhang W.Z.,Liu W.,Ding M.Z.,Li X.,Zhao G.R.,Qiao J.J.,Cheng J.S.,Zhao M.,Kuang Z.,Wang X.,Martin J.A.,Stracquadanio G.,Yang K.,Bai X.,Zhao J.,Hu M.L.,Lin Q.H.,Zhang W.Q.,Shen M.H.,Chen S.,Su W.,Wang E.X.,Guo R.,Zhai F.,Guo X.J.,Du H.X.,Zhu J.Q.,Song T.Q.,Dai J.J.,Li F.F.,Jiang G.Z.,Han S.L.,Liu S.Y.,Yu Z.C.,Yang X.N.,Chen K.,Hu C.,Li D.S.,Jia N.,Liu Y.,Wang L.T.,Wang S.,Wei X.T.,Fu M.Q.,Qu L.M.,Xin S.Y.,Liu T.,Tian K.R.,Li X.N.,Zhang J.H.,Song L.X.,Liu J.G.,Lv J.F.,Xu H.,Tao R.,Wang Y.,Zhang T.T.,Deng Y.X.,Wang Y.R.,Li T.,Ye G.X.,Xu X.R.,Xia Z.B.,Zhang W.,Yang S.L.,Liu Y.L.,Ding W.Q.,Liu Z.N.,Zhu J.Q.,Liu N.Z.,Walker R.,Luo Y.,Wang Y.,Shen Y.,Yang H.,Cai Y.,Ma P.S.,Zhang C.T.,Bader J.S.,Boeke J.D.,and Yuan Y.J.,2017,"Perfect"designer chromosome V and behavior of a ring derivative,Science,(10):355
Yin J.,Li X.,Zhan Y.,Li Y.,Qu Z.,Sun L.,Wang S.,Yang J.,and Xiao J.,2017,Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch,BMC Plant Biology,17:214