摘要
根据GenBank登录的蒜氨酸酶序列(S73324)设计上下游引物,利用RT-PCR技术从浙江大蒜鳞茎中扩增蒜氨酸酶基因,获得了长度为1523bp的cDNA序列,提交GenBank(登录号:FJ786257,命名为浙江蒜氨酸酶)。利用生物信息学相关软件对该序列进行同源性比较、进化分析、蛋白质的结构和酶活性中心预测,引用pPICZaC载体构建蒜氨酸酶毕赤酵母真核表达重组质粒。结果表明:浙江蒜氨酸酶与其他葱属植物如大蒜、洋葱、冬葱、火葱和细香葱蒜氨酸酶具有高度同源性,进化关系比较接近,目的基因可以成功构建到毕赤酵母表达载体。浙江蒜氨酸酶三维结构呈树枝状,蛋白质分子的N端含有一个类似表皮生长因子结构域,酶的中心含一个天冬氨酸氨基转移酶超家族结构域;蛋白质结构中Lys-277可能是磷酸吡哆醛的结合位点,Lys-277周围区域可能是酶活性中心部位。重组质粒经鉴定构建正确。
Primers were designed based on the reported sequences of alliinase（S73324）in GenBank.The cDNA gene encoding alliinase protein was extracted from Zhejiang garlic bulb by reverse transcription polymerase chain reaction（RT-PCR）.PCR product was about 1 523 bp DNA segment.The sequence was submitted to GenBank（accession number：FJ786257）,named Zhejiang alliinase（ZJ alliinase）.ZJ alliinase was compared with the other alliinases reported in aspect of homology and evolution,the protein structure and the enzyme active center were also predicted by bioinformatics softwares.The Alliinase gene was cloned to the pPICZaC vector to construct the Pichia pastoris expression plasmid.The results showed the high similarity and approximation to those sequences of alliinase such as garlic,onion,scallion,allium ascalonicum and chive,the target gene was successfully inserted to the pPICZaC vector.The three dimensional structure of ZJ alliinase was arborization.A EGF-like domain was in the ZJ alliinase N-terminal,and an aspartate aminotransferase（AAT）superfamily domain was in its center.The amino acid residue Lys-277 could be the combining site of pyridoxal phosphate,and the sequence around it could be the potential enzyme active center.The pPICZaC-alliinase recombination plasmid was completely accurate by using restrictional enzyme assay,plasmid PCR identification and recombinant plasmid sequence analysis.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2010年第3期258-263,283,共7页
Journal of Jilin Agricultural University
基金
浙江省教育厅资助项目(Y200805242)
浙江中医药大学校级重点资助项目(2009ZZ05)
关键词
蒜氨酸酶
大蒜
克隆
序列分析
毕赤酵母
alliinase
garlic
cloning
sequence analysis
Pichia pastoris
