乙酰辅酶A羧化酶CT功能域基因的克隆与表达
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摘要
乙酰辅酶A羧化酶(简称ACCase)是脂肪酸代谢中的关键酶,在生物体内催化乙酰辅酶A生成丙二酸单酰辅酶A的反应,这是脂肪酸从头合成的第一步也是限速步骤。乙酰辅酶A羧化酶属于生物素依赖型酶,发挥作用时需要生物素作为辅基。近年来发现,禾本科植物中的乙酰辅酶A羧化酶其羧基转移酶(CT)功能域是两类除草剂作用的靶标,因此ACCase CT功能域已经成为研究热点。
本研究主要分为两部分。第一部分克隆了中国春小麦质体中乙酰辅酶A羧化酶CT功能域基因截短序列,并且构建其重组质粒pET28a+-CT2.1kb和pET28a+-CT1.9kb,之后在大肠杆菌中诱导表达。实验证明重组质粒pET28a+-CT2.1kb可以在大肠杆菌中大量表达,但是表达产物为包涵体,在对诱导表达条件进行优化后,能得到一些可溶的目的蛋白;重组质粒pET28a+-CT1.9kb不能在大肠杆菌中表达。
第二部分克隆酿酒酵母中乙酰辅酶A羧化酶CT功能域的基因,并构建了其在毕赤酵母中表达的重组质粒pPIC9K-CT,随后在毕赤酵母GS115中进行分泌表达。实验证明重组质粒pPIC9K-CT能够在毕赤酵母中表达,并对该重组蛋白进行纯化及性质鉴定。
通过基因工程方法实现了中国春小麦质体中的乙酰辅酶A羧化酶CT功能域基因截短序列的克隆表达;以及酿酒酵母中的乙酰辅酶A羧化酶CT功能域基因在毕赤酵母中的可溶性表达。对研究除草剂与CT功能域相互作用的分子机制,以及筛选新的除草剂等方面有重要的应用价值。
Acetyl-CoA carboxylases are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT)domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. People have determined the crystal structures of the CT domain of yeast ACCase in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain.
ACCases have been found in most living organisms. There are mainly two kinds of ACCases, including procaryotic enzyme and eukaryotic enzyme. Multi-subunit ACCase, while multi-domain ACCase mainly in humans and most other eukaryotes.
The former is composed of four subunits, biotin carboxyl carrier protein, biotin carboxylase, carboxyltransferaseαandβ. The later is composed of a single strand peptide including four function domains, BC, BCCP, CT-αand CT-β.
In plant, there are two kinds of ACCases. Gramineae ACCases both in plastid and in cytosol are multi-domain eukaryotic ACCases. It has been found that Gramineae ACCase in plastid is the target of two classes of herbicides, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs). These herbicides selectively inhibit the CT activity of ACCases and block the growth of gramineae grasses. Several grass species are tolerant of APP and CHD herbicides based on the presence of an insensitive form of ACCase because these herbicides are widely used. The molecular recognition mechanism between CT domain of ACCase and inhibitors is not known. In order to systemically investigate these problems, it is necessary to get purified CT domain protein of ACCase.
Recently Tong L. and his companions have successfully overexpressed the homomeric ACCase CT domain of yeast in E.coli expression system. However the expression of ACCase CT domain from plant plastids has not been reported. But the homology and phylogenetic tree show that ACCase in yeast is more similar to that of wheat plastid other than to that of wheat cytosol. So we confirmed it is feasible to express CT domain of wheat plastid in vitro.
Exogenous gene's expression in E.Coli including two influencing factor that is copies and translates. There are three types according to the way of expression: First, expresses in the cytosol; Second, fusion expression with other mycelium protein; Third, adds the signal peptide to the N terminatio, which can carry on the secretion expression. And the first way of expression is high efficiency, therefore this experiment uses this kind of expression. But the there are defects of the expression way that is the expressing product to be very easy to gather and forms inclusion body. Many kinds of factors influence the formation of inclusion body, such as protein’s nature, yield condition (host fungus, raise temperature, pH of the culture medium and so on), expression efficiency and molecular chaperones and so on.
The front part of this research is expression of truncating ACCase CT gene from Chinese spring wheat plastid. Firstly constructed recombinant plasmid T-CT2.1kb and T-CT1.9kb of Chinese spring wheat plastid ACCase CT gene truncating sequence. Our group has already constructed the pET28a+-CT2.3kb recombinant plasmid which includes Chinese spring wheat plastid ACCase CT’s total length sequence. With this recombinant plasmid as template, we obtains massive gene of Chinese spring wheat plastid ACCase CT gene truncating sequence by Polymerase Chain Reaction, and clones this gene to the PMD18-T vector, in order to enhance the accuracy of clone .
Constructs expression recombinant plasmid pET28a+-CT1.9kb and pET28a+-CT2.1kb of Chinese spring wheat plastid ACCase CT gene’s truncating sequence. afterward expresses these two kind of proteins massively in E.coli. And simultaneously to obtain the high expression quantity and is also the soluble goal protein, has carried on some optimizations to the expression condition. The best expression condition is in 16℃the induction, derivative IPTG is divided two flowing to join or joins the sorbitol in the culture medium, expresses the fungus to use BLP, and the expression vector is pET32a+.
Which the system uses is one kind of shuttle material particle, the goal gene completes connection to the vector in E.coli, then transforms into the yeast expression strain GS115 and to carry on the reorganization with its genome team. The conformity way has two kinds: one kind is the carrier linearization, forms the dissociation Aox15 the `end and 3 `ends, has the homologous double exchange with the host chromosome, in the substitution host chromosome Aox1 code area. Because the host chromosome's Aox1 structural gene is deleted, cannot code the Aox1 enzyme, therefore obtains the Muts phenotype converter. Another kind is the carrier linearization, but does not produce the dissociation 5 `end and 3 `ends, has the list exchange conformity with the host chromosome.
The present paper's second part is secretion expression of Saccharomyces cerevisia yeast ACCase the CT in the Pichia yeast GS115 strain. Construction recombinant plasmid pPIC9K-CT of Saccharomyces cerevisia yeast ACCase CT to expression this protein, afterward this recombinant plasmid is transformed into yeast expression strain GS115. Using has the double exchange reorganization sub-phenotype is His+Muts characteristic screening masculine gender clone, that is these clones may not grow the mellow oxydases gene (Aox1) flaw including in the histidine culture medium to cause the yeast to be slow-growing. The masculine clone carries on the induction to express, has carried on the optimization to the culture medium pH value and the induction expression time. The best pH value is 8.0, inducing expression time is 3 days.
本研究主要分为两部分。第一部分克隆了中国春小麦质体中乙酰辅酶A羧化酶CT功能域基因截短序列,并且构建其重组质粒pET28a+-CT2.1kb和pET28a+-CT1.9kb,之后在大肠杆菌中诱导表达。实验证明重组质粒pET28a+-CT2.1kb可以在大肠杆菌中大量表达,但是表达产物为包涵体,在对诱导表达条件进行优化后,能得到一些可溶的目的蛋白;重组质粒pET28a+-CT1.9kb不能在大肠杆菌中表达。
第二部分克隆酿酒酵母中乙酰辅酶A羧化酶CT功能域的基因,并构建了其在毕赤酵母中表达的重组质粒pPIC9K-CT,随后在毕赤酵母GS115中进行分泌表达。实验证明重组质粒pPIC9K-CT能够在毕赤酵母中表达,并对该重组蛋白进行纯化及性质鉴定。
通过基因工程方法实现了中国春小麦质体中的乙酰辅酶A羧化酶CT功能域基因截短序列的克隆表达;以及酿酒酵母中的乙酰辅酶A羧化酶CT功能域基因在毕赤酵母中的可溶性表达。对研究除草剂与CT功能域相互作用的分子机制,以及筛选新的除草剂等方面有重要的应用价值。
Acetyl-CoA carboxylases are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases. The carboxyltransferase (CT)domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim. People have determined the crystal structures of the CT domain of yeast ACCase in complex with the herbicide haloxyfop or diclofop. The inhibitors are bound in the active site, at the interface of the dimer of the CT domain.
ACCases have been found in most living organisms. There are mainly two kinds of ACCases, including procaryotic enzyme and eukaryotic enzyme. Multi-subunit ACCase, while multi-domain ACCase mainly in humans and most other eukaryotes.
The former is composed of four subunits, biotin carboxyl carrier protein, biotin carboxylase, carboxyltransferaseαandβ. The later is composed of a single strand peptide including four function domains, BC, BCCP, CT-αand CT-β.
In plant, there are two kinds of ACCases. Gramineae ACCases both in plastid and in cytosol are multi-domain eukaryotic ACCases. It has been found that Gramineae ACCase in plastid is the target of two classes of herbicides, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs). These herbicides selectively inhibit the CT activity of ACCases and block the growth of gramineae grasses. Several grass species are tolerant of APP and CHD herbicides based on the presence of an insensitive form of ACCase because these herbicides are widely used. The molecular recognition mechanism between CT domain of ACCase and inhibitors is not known. In order to systemically investigate these problems, it is necessary to get purified CT domain protein of ACCase.
Recently Tong L. and his companions have successfully overexpressed the homomeric ACCase CT domain of yeast in E.coli expression system. However the expression of ACCase CT domain from plant plastids has not been reported. But the homology and phylogenetic tree show that ACCase in yeast is more similar to that of wheat plastid other than to that of wheat cytosol. So we confirmed it is feasible to express CT domain of wheat plastid in vitro.
Exogenous gene's expression in E.Coli including two influencing factor that is copies and translates. There are three types according to the way of expression: First, expresses in the cytosol; Second, fusion expression with other mycelium protein; Third, adds the signal peptide to the N terminatio, which can carry on the secretion expression. And the first way of expression is high efficiency, therefore this experiment uses this kind of expression. But the there are defects of the expression way that is the expressing product to be very easy to gather and forms inclusion body. Many kinds of factors influence the formation of inclusion body, such as protein’s nature, yield condition (host fungus, raise temperature, pH of the culture medium and so on), expression efficiency and molecular chaperones and so on.
The front part of this research is expression of truncating ACCase CT gene from Chinese spring wheat plastid. Firstly constructed recombinant plasmid T-CT2.1kb and T-CT1.9kb of Chinese spring wheat plastid ACCase CT gene truncating sequence. Our group has already constructed the pET28a+-CT2.3kb recombinant plasmid which includes Chinese spring wheat plastid ACCase CT’s total length sequence. With this recombinant plasmid as template, we obtains massive gene of Chinese spring wheat plastid ACCase CT gene truncating sequence by Polymerase Chain Reaction, and clones this gene to the PMD18-T vector, in order to enhance the accuracy of clone .
Constructs expression recombinant plasmid pET28a+-CT1.9kb and pET28a+-CT2.1kb of Chinese spring wheat plastid ACCase CT gene’s truncating sequence. afterward expresses these two kind of proteins massively in E.coli. And simultaneously to obtain the high expression quantity and is also the soluble goal protein, has carried on some optimizations to the expression condition. The best expression condition is in 16℃the induction, derivative IPTG is divided two flowing to join or joins the sorbitol in the culture medium, expresses the fungus to use BLP, and the expression vector is pET32a+.
Which the system uses is one kind of shuttle material particle, the goal gene completes connection to the vector in E.coli, then transforms into the yeast expression strain GS115 and to carry on the reorganization with its genome team. The conformity way has two kinds: one kind is the carrier linearization, forms the dissociation Aox15 the `end and 3 `ends, has the homologous double exchange with the host chromosome, in the substitution host chromosome Aox1 code area. Because the host chromosome's Aox1 structural gene is deleted, cannot code the Aox1 enzyme, therefore obtains the Muts phenotype converter. Another kind is the carrier linearization, but does not produce the dissociation 5 `end and 3 `ends, has the list exchange conformity with the host chromosome.
The present paper's second part is secretion expression of Saccharomyces cerevisia yeast ACCase the CT in the Pichia yeast GS115 strain. Construction recombinant plasmid pPIC9K-CT of Saccharomyces cerevisia yeast ACCase CT to expression this protein, afterward this recombinant plasmid is transformed into yeast expression strain GS115. Using has the double exchange reorganization sub-phenotype is His+Muts characteristic screening masculine gender clone, that is these clones may not grow the mellow oxydases gene (Aox1) flaw including in the histidine culture medium to cause the yeast to be slow-growing. The masculine clone carries on the induction to express, has carried on the optimization to the culture medium pH value and the induction expression time. The best pH value is 8.0, inducing expression time is 3 days.
引文
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[2]Barber M C, Price N T, Travers M T. Structure and regulation of acetyl-CoA carboxylase genes of metazoa [J].Biochim. Biophys.Acta,2005,1733:1–28.
[3]Freiberg C, Fischer H P, Brunner N A. Discovering the mechanisms of action of novel antibacterial agents through transcriptional profiling of conditional mutants [J].Antimicrob Agents Chemother,2005,49:749–759.
[4]Pohlmann J, Lampe T, Shimada M, Nell P G,Pernerstorfer J, Svenstrup N, et al. Pyrrolidinedione derivatives as antibacterial agents with a novel mode of action [J].Bioorg. Med. Chem. Lett,2005,15:1189–1192.
[5] Harwood H J Jr. Acetyl-CoA carboxylase inhibition for the treatment of metabolic syndrome [J].Curr. Opin. Investig.Drugs,2004,5:283–289.
[6]Sasaki Y, Nagano Y. Plant acetyl-CoA carboxylase:structure, biosynthesis, regulation and gene manipulation for plant breeding [J].Biosci. Biotechnol. Biochem, 2004,68:1175–1184.
[7]Shen Y, Volrath S L, Weatherly S C, Elich T D,Tong L. A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product [J].Mol.Cell,2004,16:881–891.
[8]Reszko A E, Kasumov T, David F, Thomas K. R., Jobbins K A, Cheng J F, et al. Regulation of malonyl-CoA concentration and turnover in the normal heart [J].Biol. Chem,2004,279:34298–34301.
[9]Hoja U, Marthol S, Hofmann J, Stegner S, Schulz R, Meier S, et al. HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae [J]. Biol.Chem,2004,279:21779–21786.
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