富含蛋氨酸醇溶蛋白在产朊假丝酵母中的表达研究
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摘要
为构建表达富含蛋氨酸醇溶蛋白的产朊假丝酵母工程菌株,利用基因工程技术,将玉米δ-10kD-醇溶蛋白基因(zein),酵母的GAP启动子(GAP-p)、终止子(GAP-t)拼接合成表达框,以18S rDNA片段为整合介导区,用放线菌酮(CYH)抗性基因作为筛选标记,结合pBR322质粒构建了同源整合表达载体,把zein基因同源重组整合到产朊假丝酵母染色体上,构建表达zein基因的产朊假丝酵母工程菌株。蛋氨酸含量测定结果显示,重组菌株的蛋氨酸表达量比起始菌株提高了17.14%。该产朊假丝酵母工程菌的构建为下一步提高其蛋氨酸产量的研究和应用打下了良好的基础。
The present study was designed to construct a genetically engineered strain of Candida utilis that expresses methionine-rich zein protein. By using genetic engineering method, a methionine-rich δ-10 kD-zein gene of maize endosperm,the promoter(GAP-p) gene and the terminator(GAP-t) gene from yeast were spliced into an expression cassette; subsequently,a homologous recombinant expression vector was constructed with a 18 S r DNA fragment(serving as an integrated mediated region), a cycloheximide(CYH) resistance gene(serving as a selection marker) and the pBR322 plasmid; the zein gene was integrated into the chromosome of Candida utilis by using homologous recombination. The determination results of methionine content by HPLC demonstrated that the expression of methionine in the recombinant strain was increased by 17.14% than the wild one. The constructed genetically engineered strain of Candida utilis provided technological foundation for further study on increasing its production of methionine.
The present study was designed to construct a genetically engineered strain of Candida utilis that expresses methionine-rich zein protein. By using genetic engineering method, a methionine-rich δ-10 kD-zein gene of maize endosperm,the promoter(GAP-p) gene and the terminator(GAP-t) gene from yeast were spliced into an expression cassette; subsequently,a homologous recombinant expression vector was constructed with a 18 S r DNA fragment(serving as an integrated mediated region), a cycloheximide(CYH) resistance gene(serving as a selection marker) and the pBR322 plasmid; the zein gene was integrated into the chromosome of Candida utilis by using homologous recombination. The determination results of methionine content by HPLC demonstrated that the expression of methionine in the recombinant strain was increased by 17.14% than the wild one. The constructed genetically engineered strain of Candida utilis provided technological foundation for further study on increasing its production of methionine.
引文
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[2]张成喜,滕乐帮,刘晓,等.过瘤胃蛋氨酸和肉桂醛对奶牛瘤胃微生物蛋白产量和养分消化率的影响[J].中国畜牧杂志,2017,53(9):92-96.
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[6]韩兆玉,周岩民,王根林,等.过瘤胃蛋氨酸对泌乳中期奶牛产奶量以及乳成分的影响[J].草食家畜,2004(3):43-45.
[7]甘佳,付茂忠,王巍,等.日粮中添加过瘤胃脂肪和过瘤胃蛋氨酸对荷斯坦奶牛生产性能的影响[J].四川畜牧兽医,2017(11):32-34.
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[9]李明华,郭亮,金曙光.全混合日粮中添加包被氨基酸对奶牛产奶量和乳成分的影响[J].中国畜牧兽医,2006,33(12):35-37.
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[11]邹阿玲,孙国军,李明强,等.过瘤胃蛋氨酸对泌乳早期奶牛生产性能的影响[J].中国奶牛,2005(2):27-29.
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[13]董贤文,左福元.过瘤胃蛋氨酸的研究与应用[J].饲料广角,2012(8):30-32.
[14]刘庆国,潘艳青,汪琨,等.产朊假丝酵母(Candida utilis)基因工程研究进展[J].工业微生物,2017,47(4):66-71.
[15] KONDO K, S AITO T,KAJIWARA S,et al. A transformation system for the yeast Candida utilis:use of a modified endogenous ribosomal DNA as a drugresistant marker and ribosomal DNA as an integration target for vector DNA[J] Journal of Bacteriology,1995,177(24):7171-7177.
[16] ZHANG A L,LUO J X,ZHANG T Y,et al. Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter[J].Aata Genetica Sinica, 2004,31(6):552-557.
[17]刘向勇,沈煜,郭亭,等.rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109.
[18]庹德财,沈文涛,言普,等.一种E.coli-Free的酵母同源重组系统稳定快速构建PLDMV侵染性克隆的新方法[J].热带作物学报,2017,38(8):1492-1500.
[19] KUNIGO M,BUERTH C,TIELKER D,et al.Heterologous protein secretion by Candida utilis[J].Applied Microbiology and Biotechnology,2013,97(16):7357-7368.
[20] HANA BONKOVA,MICHAELA OSADSKA,JAN KRAHULEC,et al. Upstream regulatory regions controlling the expression of the Candida utilis maltase gene[J].Journal of Biotechnology,2014(189):136-142.
[21] BAGGA S, ARMEND ARIS A, KLYPIN A N,et al.Genetic engineering ruminal stable high methionine protein in the foliage of alfalfa[J].Plant Science,2004,166(2):273-283.
[22] WOO Y M,HU D W,LARKINS B A,et al. Genomics analysis of genes expressed in maize endospermidentifies novel seed proteins and clarifies patterns of zein gene expression[J].Plant, 2001,13(10):2297-2317.
[23] ZHANG Y,DARLINGTON H,JONES H D,et al.Expression of the gamma-zein protein of maize in seeds of transgenic barley:effects on grain composition and properties[J].Theoretical and Applied Genetics,2003,106(6):1139-1146.
[24]王广立,唐茂芝,林忠平.富硫蛋白基因对牧草百脉根的转化[J].植物学报,1994,36(3):204-208.
[25]李雷,刘松梅,胡鸢雷,等.导入玉米10KD醇溶蛋白基因提高马铃薯块茎中含硫氨基酸的含量[J].科学通报,2000,45(12):1313-1317.
[26]刘晓颍.农杆菌介导的玉米醇溶蛋白基因转化紫花苜蓿的研究[D].乌鲁木齐:新疆大学,2007.
[27]韩小花,岳润清,郭书磊,等.玉米品质基因聚合对籽粒氨基酸组分及醇溶蛋白的影响[J].玉米科学,2018,26(2):53-57.
[28]娄丽,高学军.富含蛋氨酸玉米醇溶蛋白在大肠杆菌中的表达[J].生物技术,2011,21(5):27-30.
[29]娄丽,高学军,敖金霞.富含蛋氨酸玉米醇溶蛋白在枯草芽胞杆菌中的表达[J].微生物学杂志,2011,31(5):31-34.
[30]杨红兰,王炜,包慧芳,等.产朊假丝酵母整合表达载体的构建[J].微生物学报,2009,49(3):316-323.
[31] TAMAKAWA H,IKUSHIMA S, YOSHIDA S.Construction of a Candida utilis strain with ratiooptimized expression of xylose metabolizing enzyme genes by cocktail multicopy integration method[J].Journal of Bioscience and Bioengineering,2013,115(5):532-539.
[32] TAMAKAWA H,MITA T,YOKOYAMA A,et al.Metabolic engineering of Candida utilis for isopropanol production[J].Applied Microbiology and Biotechnology,2013,97(14):6231-6239.
[2]张成喜,滕乐帮,刘晓,等.过瘤胃蛋氨酸和肉桂醛对奶牛瘤胃微生物蛋白产量和养分消化率的影响[J].中国畜牧杂志,2017,53(9):92-96.
[3] ROBERT J C,SLOAN B K,DENIS C. The effect of graded amounts of rumen-protected methionine on lactational responses in dairy cows[J].Journal of Dairy Science, 1996,79(1):256.
[4] XU S,HARRISON J H,CHALUPA W,et al. The effect of ruminal bypass lysine and methionine on milk yield and composition of lactating cows[J].Journal of Dairy Science, 1998,81(4):1062-1077.
[5] MISCIATTELLI L,KRISTENSEN V F,VESTERGAARDM,et al. Milk production, nutrient utilization, and endocrine responses to increased postruminal lysine and methionine supply in dairy cows[J].Journal of Dairy Science, 2003,86(1):275-286.
[6]韩兆玉,周岩民,王根林,等.过瘤胃蛋氨酸对泌乳中期奶牛产奶量以及乳成分的影响[J].草食家畜,2004(3):43-45.
[7]甘佳,付茂忠,王巍,等.日粮中添加过瘤胃脂肪和过瘤胃蛋氨酸对荷斯坦奶牛生产性能的影响[J].四川畜牧兽医,2017(11):32-34.
[8] SOCHA M T, PUTNAM D E, GARTH WAITE B D,et al. Improving intestinal amino acid supply of pre-and postpartum dairy cows with rumen-protected methionine and lysine[J].Journal of Dairy Science,2005,88(3):1113-1126.
[9]李明华,郭亮,金曙光.全混合日粮中添加包被氨基酸对奶牛产奶量和乳成分的影响[J].中国畜牧兽医,2006,33(12):35-37.
[10] KROBER T F,KREUZER M,SENN M,et al. Effects of rumen-protected methionine in a low protein ration on metabolic traits and performance of early lactating cows as opposed to rations with elevated crude protein content[J].Journal of Animal Physiology and Animal Nutrition, 2010,84(5):148-164.
[11]邹阿玲,孙国军,李明强,等.过瘤胃蛋氨酸对泌乳早期奶牛生产性能的影响[J].中国奶牛,2005(2):27-29.
[12] GIRARD C L,LAPIERRE H,MATTE J J,et al. Effects of dietary supplements of folic acid and rumenprotected methionine on lactational performance and folate metabolism of dairy cows[J].Journal of DairyScience,2005,88(2):660-670.
[13]董贤文,左福元.过瘤胃蛋氨酸的研究与应用[J].饲料广角,2012(8):30-32.
[14]刘庆国,潘艳青,汪琨,等.产朊假丝酵母(Candida utilis)基因工程研究进展[J].工业微生物,2017,47(4):66-71.
[15] KONDO K, S AITO T,KAJIWARA S,et al. A transformation system for the yeast Candida utilis:use of a modified endogenous ribosomal DNA as a drugresistant marker and ribosomal DNA as an integration target for vector DNA[J] Journal of Bacteriology,1995,177(24):7171-7177.
[16] ZHANG A L,LUO J X,ZHANG T Y,et al. Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter[J].Aata Genetica Sinica, 2004,31(6):552-557.
[17]刘向勇,沈煜,郭亭,等.rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用[J].山东大学学报,2005,40(3):105-109.
[18]庹德财,沈文涛,言普,等.一种E.coli-Free的酵母同源重组系统稳定快速构建PLDMV侵染性克隆的新方法[J].热带作物学报,2017,38(8):1492-1500.
[19] KUNIGO M,BUERTH C,TIELKER D,et al.Heterologous protein secretion by Candida utilis[J].Applied Microbiology and Biotechnology,2013,97(16):7357-7368.
[20] HANA BONKOVA,MICHAELA OSADSKA,JAN KRAHULEC,et al. Upstream regulatory regions controlling the expression of the Candida utilis maltase gene[J].Journal of Biotechnology,2014(189):136-142.
[21] BAGGA S, ARMEND ARIS A, KLYPIN A N,et al.Genetic engineering ruminal stable high methionine protein in the foliage of alfalfa[J].Plant Science,2004,166(2):273-283.
[22] WOO Y M,HU D W,LARKINS B A,et al. Genomics analysis of genes expressed in maize endospermidentifies novel seed proteins and clarifies patterns of zein gene expression[J].Plant, 2001,13(10):2297-2317.
[23] ZHANG Y,DARLINGTON H,JONES H D,et al.Expression of the gamma-zein protein of maize in seeds of transgenic barley:effects on grain composition and properties[J].Theoretical and Applied Genetics,2003,106(6):1139-1146.
[24]王广立,唐茂芝,林忠平.富硫蛋白基因对牧草百脉根的转化[J].植物学报,1994,36(3):204-208.
[25]李雷,刘松梅,胡鸢雷,等.导入玉米10KD醇溶蛋白基因提高马铃薯块茎中含硫氨基酸的含量[J].科学通报,2000,45(12):1313-1317.
[26]刘晓颍.农杆菌介导的玉米醇溶蛋白基因转化紫花苜蓿的研究[D].乌鲁木齐:新疆大学,2007.
[27]韩小花,岳润清,郭书磊,等.玉米品质基因聚合对籽粒氨基酸组分及醇溶蛋白的影响[J].玉米科学,2018,26(2):53-57.
[28]娄丽,高学军.富含蛋氨酸玉米醇溶蛋白在大肠杆菌中的表达[J].生物技术,2011,21(5):27-30.
[29]娄丽,高学军,敖金霞.富含蛋氨酸玉米醇溶蛋白在枯草芽胞杆菌中的表达[J].微生物学杂志,2011,31(5):31-34.
[30]杨红兰,王炜,包慧芳,等.产朊假丝酵母整合表达载体的构建[J].微生物学报,2009,49(3):316-323.
[31] TAMAKAWA H,IKUSHIMA S, YOSHIDA S.Construction of a Candida utilis strain with ratiooptimized expression of xylose metabolizing enzyme genes by cocktail multicopy integration method[J].Journal of Bioscience and Bioengineering,2013,115(5):532-539.
[32] TAMAKAWA H,MITA T,YOKOYAMA A,et al.Metabolic engineering of Candida utilis for isopropanol production[J].Applied Microbiology and Biotechnology,2013,97(14):6231-6239.