论文部分内容阅读
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。
Using cDNA-AFLP technique, we isolated a differential fragment related to the mature senescence of papaya fruits. Bioinformatics analysis confirmed that the fragment belongs to the cysteine protease (CP) gene. The differential fragment sequence was searched in the papaya genome sequence in NCBI to obtain the cysteine protease gene DNA sequence. The open reading frame was designed. The full-length cDNA of this gene was amplified by RT-PCR from papaya flesh RNA and named as CpCP (Accession No. JN689334). This gene belongs to the C1A subfamily of the C1 peptidase family and codes for 471 amino acids and contains a family-specific catalytic triad: Cys166-His302-Asn322. Real-time PCR results showed that CpCP was abundantly expressed in fruits, which was several times as high as that in roots, stems, leaves and flowers. CpCP was induced by ethylene and inhibited by 1-MCP, The same process, indicating that CpCP involved in the ripe papaya fruit aging process.