番木瓜半胱氨酸蛋白酶基因CpCP的分离及表达分析

Cloning and Expression Analysis of Cysteine Protease Gene CpCP from Papaya

采用cDNA-AFLP技术分离了一个与番木瓜果实成熟衰老相关的差异片段,经生物信息学分析证实该片段属于半胱氨酸蛋白酶(Cysteine Protease,CP)基因。将差异片段序列在NCBI中的番木瓜基因组序列中搜索,获得了半胱氨酸蛋白酶基因DNA序列。设计开放型阅读框上、下游引物,以木瓜果肉RNA逆转录的cDNA为模板扩增该基因全长cDNA序列,命名为CpCP(登录号:JN689334)。该基因属于C1肽酶家族中的C1A亚家族,编码471个氨基酸,含有家族特有的催化三联体:Cys166-His302-Asn322。Real-time PCR结果显示CpCP在果实中大量表达,是根、茎、叶、花中表达量的几十倍;CpCP受乙烯处理诱导,受1-MCP处理抑制,表达模式与番木瓜果实成熟衰老进程一致,说明CpCP参与了番木瓜果实成熟衰老进程。

A polymorphic fragment associated with fruit ripening in papaya was obtained by c DNA-AFLP analysis and identified as a CP（Cysteine Protease）gene according to bioinformatic analysis. The sequence of open reading frame of CP was obtained from reference papaya genomic sequence in NCBI using the polymorphic fragment as a query. The full-length c DNA of CP was isolated from fruit（primers were designed according to open reading frame of CP）,and the cloned gene was designated as CpCP（Gen Bank No.：JN689334）. Using bioinformatic analysis,this gene was identified as a member of the C1 A gene subfamily of C1 Peptidase family,which encodes 471 amino acids and has active sites of Cys166-His302-Asn322. Real-time PCR analysis revealed that the expression level in fruits was the greatest,which has dozens of times than that in root,stem,leaf,and flower. In addition,the expression level was highly induced by ethylene and inhibited by 1-MCP. The expression pattern is consistent with the fruit senescence process,indicating that CpCP is involved in the fruit senescence in papaya.