| mleA基因在酿酒酵母中的整合型表达 |
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| 引用本文： | 刘延琳,李华.mleA基因在酿酒酵母中的整合型表达[J].中国农业科学,2009,42(4):1372-1377. |
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| 作者姓名： | 刘延琳 李华 |
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| 作者单位： | 西北农林科技大学葡萄酒学院/陕西省葡萄-葡萄酒工程中心,陕西,杨凌,712100 |
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| 基金项目： | 西北农林科技大学人才计划，国家高技术研究发展计划(863计划)，国家葡萄产业技术体系建设专项经费 |
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| 摘 要： | 【目的】进行中国自行筛选的专利菌种酒酒球菌 SD-2a（Oenococcus oeni SD-2a）的苹果酸－乳酸酶基因在酿酒酵母中的整合型表达，使葡萄酒生产过程中的酒精发酵和苹果酸－乳酸发酵（malolactic fermentation，MLF）同时进行。【方法】克隆Oenococcus oeni SD-2a的苹果酸－乳酸酶基因mleA，以PGK1强启动子和ADH1终止子为调控元件，以酵母整合型质粒YIp5为载体，构建重组表达质粒pYILmleA，并转化酿酒酵母（Saccharomyces cerevisiae）YS59，经筛选鉴定获得酵母转化子YS59/pYILmleA。进行转化子的营养缺陷型和交配型鉴定、菌落PCR鉴定、SDS-PAGE检测及斑点杂交检测，并对酵母转化子培养上清液进行L-苹果酸及L-乳酸含量的HPLC分析，检测目的基因的功能性表达。【结果】转化子的营养缺陷型和交配型鉴定、菌落PCR鉴定表明获得了阳性转化子;SDS-PAGE检测表明获得的转化子表达了目标蛋白，斑点杂交检测表明目的基因整合到受体菌中。模拟发酵表明mleA基因整合到受体菌中后进行了功能性表达，将培养基中的L-苹果酸转化成L-乳酸，使得培养液中的L-苹果酸含量极显著降低。转化子YS59/pYILmleA在添加5 648 mg?L-1 L-苹果酸和10%葡萄糖的SD/-Ura培养基中培养4 d，培养液上清中L-苹果酸的剩余含量与空载体转化子YS59/YIp5（对照）差异极显著，苹果酸的相对降低率为20.18%～20.85%。供试的转化子L-乳酸生成量为1 278～1 312 mg?L-1，对照未检出乳酸的生成。【结论】中国自主筛选的专利菌种O.oeni SD-2a的mleA基因的整合型表达质粒构建成功并在酿酒酵母中进行了功能性表达。
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| 关 键 词： | 酒酒球菌 苹果酸－乳酸酶基因 整合重组表达 酿酒酵母 |
| 收稿时间： | 2008-7-30 |
Integrated Expression of mleA Gene in Saccharomyces cerevisiae |
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| LIU Yan-lin,LI Hua.Integrated Expression of mleA Gene in Saccharomyces cerevisiae[J].Scientia Agricultura Sinica,2009,42(4):1372-1377. |
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| Authors: | LIU Yan-lin LI Hua |
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| Institution: | College of Enology, Northwest Sci-Tech University of Agriculture and Forestry/Shaanxi Engineering Research Center for Viti-viniculture |
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| Abstract: | 【Objective】 In this paper, researches concerning the malolactic enzyme gene mleA cloning from a patent strain O. oeni SD-2a screened from Chinese wine and integrated expressing in S. cerevisiae were made in order to perform alcoholic fermentation (AF) and malolactic fermentation (MLF) simultaneously during winemaking. 【Method】 Cloned malolactic enzyme gene mleA from Oenococcus oeni SD-2a, PGK1 promoter and ADH1 terminator were ligated and inserted into integrating vector YIp5 to construct the expression plasmid named pYILmleA. When transformed into S. cerevisiae YS59, the resulted yeast transformants YS59/pYILmleA were screened on SD/-Ura and identified by auxotrophic test, mating type test and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot blotting hybridization. The transformants were cultured in medium containing L-malate and the culture supernatants were collected and L-malate and L-lactic acid content were detected by HPLC to confirm if functional expression were achieved. 【Result】 Auxotrophic test, mating type test and colony PCR showed positive transformants were obtained. Target protein was detected by SDS-PAGE and the targeted gene was integrated into the chromosome detected by dot blotting hybridization. When the transformants of YS59/pYILmleA were cultured in SD/-Ura added with 10% glucose and 5648 mg?L-1 L-malate for 4d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC, 1278-1312 mg?L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18%-20.85%. L-malate contents and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t, test respectively. 【Conclusion】 The result indicated that the integrated expressive plasmid containing mleA gene from patent strain O.oeni SD-2a screened from Chinese wine was constructed and the functional expression was achieved in recombinants S. cerevisiae. |
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| Keywords: | Oenococcus oeni malolactic enzyme gene integrated recombinant expression Saccharomyces cerevisiae |
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