番木瓜酪氨酸氨基转移酶基因CpTAT的分离与分析

Isolation and Analysis of Tyrosine Aminotransferase(TAT) Gene in Papaya

番木瓜酪氨酸氨基转移酶(Tyrosine aminotransferase,TAT)是维生素E合成途径中的一个关键酶。在已有番木瓜果实成熟差异基因片段序列的基础上,采用RACE技术克隆了TAT基因的全长c DNA序列,命名为Cp TAT。该基因全长包含5′非编码区98 bp,3′非编码区232 bp,开放性阅读框1 266 bp,编码421个氨基酸。序列分析表明,该基因为谷草转氨酶超家族成员,催化活性位点为第253位的赖氨酸,编码蛋白与毛果杨、野草莓、桃、拟南芥和水稻的TAT蛋白同源性分别为83.69%、81.09%、80.76%、78.87%、54.67%。荧光定量分析表明,Cp TAT基因在根中的表达量最高,在茎和果实中的表达较低,在叶中的表达量最低。与对照相比,外源乙烯处理能诱导番木瓜果实中Cp TAT基因表达量升高,而1-MCP处理则抑制了该基因表达,在果实衰老期表达量升高,该基因可能在番木瓜果实成熟衰老中起作用。

Tyrosine aminotransferase（TAT） is one of the most important key enzymes in the vitamin E biosynthesis pathway.The full-length c DNA of TAT was isolated from papaya fruit using RACE-PCR and designated as Cp TAT.The primers were designed according to a fruit ripening polymorphic fragment which was obtained from c DNA-AFLP analysis.The Cp TAT c DNA sequence contains 5′-UTR of 98 bp,3′-UTRA of 232 bp,and ORF of1 266 bp.Sequencing analysis showed that the gene was a member of the aspartate aminotransferase superfamily,which encoding 421 amino acids and having an active site of Lys253.The amino acid sequence was 83.69%,81.09%,80.76%,78.87%,and 54.67% identity with TAT from Populus trichocarpa,strawberry,peach,Arabidopsis,and rice.Real-time PCR analysis revealed that the order of relative expression level of the Cp TAT gene in different papaya organs was root,stem,fruit,and leave.In addition,the Cp TAT expression was induced by ethylene,inhibited by 1-MCP,and increased in aging period,indicating that Cp TAT may be involved in the fruit senescence in papaya.