香蕉果实果胶裂解酶基因cDNA克隆及序列分析

Cloning and sequence of cDNA fragments encoding pectate lyase genes from banana fruit

利用已报道果胶裂解酶基因的保守序列设计简并引物,进行RT-PCR,得到1个大约1300bp的香蕉果胶裂解酶基因cDNA片段,命名为MA-pl。DNA序列分析表明:MA-pl片段全长1277bp,包含1个882bp的开放读码框(ORF),编码294个氨基酸;其具有所有果胶裂解酶共有的保守区域:钙协调部位(Asp72,Asp74,Asp96,andAsp100)、酶活性位点(Arg152,Pro154,Arg157)及3个重要的结构域(motifI:WVDH,motifII:DGLVDAVMGSTAITVSNNYF,motifIII:LYQRMPRCRHGYFHVVWNDY);MA-pl氨基酸序列与草莓-1、葡萄、草莓-2、拟南芥、苹果、香蕉(banana-1)的相似性分别为85.8%、74.2%、79.7%、78.6%、72.4%和71.4%。

A 1277 bp cDNA fragment encoding pectate lyase, named as MA-pl, was cloned from banana fruit by using degenerate primers designed with reference to the conserved amino acid sequences of known pectate lyases by RT-PCR. Sequence analysis showed that MA-pl contains an 882 bp ORF, encoding a predicted polypeptide of 294 amino acids. Alignment of the deduced amino acid sequence of MA-pl and other putative pectate lyases showed that MA-pl has an identity of 85.8% ,74.2%, 79.7%, 78.6%, 72.4% and 71.4% respectively to pectate lyases from strawberry-1, grape, strawberry-2, arabidopsis thaliana, apple and banana-1. The amino acid residues conserved in all pectate lyase genes involved in Ca^2＋ coordination （Asp72, Asp74, Asp96, and Aspl00） and the active site conformation （Arg152, Pro154, Arg157） were present in MA-pl. Three conserved motifs （motifⅠ： WVDH, motifⅡ： DGLVDAVMGCSTAITISNNHL, motifⅢ： LYQRMPRCRHGYFHVVW NDY） characteristic to all pectate lyases were found in MA-pl, suggesting that MA-pl highly likely encodes a pectate lyase in banana.