关键词: 番木瓜, 半胱氨酸蛋白酶, 基因克隆, cDNA-AFLP, 荧光定量PCR

Abstract:

A polymorphic fragment associated with fruit ripening in papaya was obtained by cDNA-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 cDNA of CP was isolated from fruit （primers were designed according to open reading frame of CP），and the cloned gene was designated as CpCP（GenBank No.：JN689334）. Using bioinformatic analysis，this gene was identified as a member of the C1A 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 withthe fruit senescence process，indicating that CpCP is involved in the fruit senescence in papaya.

Key words: Carica papaya, cysteine protease, gene clone, cDNA-AFLP, real-time PCR