金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究
Cloning of A Laccase Gene from Flammulina velutipes and Study on Its Expression in Pichia pastoris
张银波 姜琼 江木兰 马立新
摘 要：综合运用cDNA末端快速扩增(Rapid Amplification of cDNA Ends,RACE )和基因组步行等技术克隆到一个金针菇(Flammulina velutipes)的漆酶结构基因和其对应的全长cDNA,经测序和BLAST比对分析表明该基因属于多铜氧化酶基因家族,与已发表的漆酶基因(AF176230) 的同源性最高,在氨基酸水平为72%.该结构基因命名为glccFv,cDNA命名为lccFv,其序列提交GenBank,登录号分别为AY485826和AY450406.将lccFv的开放阅读框克隆到毕赤酵母表达载体pHBM906,转化毕赤酵母GS115且实现了分泌表达.将重组毕赤酵母GS115 (pHBM565)诱导产酶,在培养温度20℃、甲醇流加量为1.0%(V/V)的情况下,其分泌表达的LCCFv的最高酶活为0.1070 U/mL,最适反应温度为45℃,最适反应pH值为3.9,在最适反应条件下其热稳定性和pH值稳定性均较好.
关键词：cDNA末端快速扩增, 基因组步行, 漆酶基因, 毕赤酵母, 分泌表达
分类号：Q939 文献标识码：A
文章编号：0001-6209(2004)06-0775-05
基金项目：国家"863计划"(2001AA214161;2002AA227011)
作者简介：张银波(1974-),男,湖北应城人,助理研究员,硕士研究生,从事微生物基因工程方面的研究.E-mail:ybzhang04@yahoo.com.马立新,通讯作者. Tel/Fax: 86-27-88666349;E-mail: malixin9@hotmail.com
作者单位：张银波（湖北大学,分子微生物学与基因工程研究室,武汉,430062;中国农业科学院油料作物研究所,农业部油料作物遗传改良重点开放实验室,武汉,430062）
姜琼（湖北大学,分子微生物学与基因工程研究室,武汉,430062）
江木兰（中国农业科学院油料作物研究所,农业部油料作物遗传改良重点开放实验室,武汉,430062）
马立新（湖北大学,分子微生物学与基因工程研究室,武汉,430062）
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收稿日期：2004年4月23日
修稿日期：2004年6月4日
出版日期：2004年12月1日
微生物学报
ACTA MICROBIOLOGICA SINICA
2004 Vol.44 No.6 P.775-779
