麻疯树苯丙氨酸解氨酶启动子的克隆和表达载体的构建

Cloning of a Phenylalanine Ammonia-lyase Promoter from Jatropha curcas and Construction of Expression Vector

苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)是苯丙烷类代谢途径的关键酶,催化苯丙氨酸转化为肉桂酸,促进黄酮、香豆素等次生代谢物的生成。本文根据已克隆的麻疯树苯丙氨酸解氨酶基因JcPAL的序列设计引物,通过DNA步移技术,克隆出长度为1334bp的JcPAL基因起始密码子上游序列。序列分析显示其不仅具备CAAT、TATA盒这些保守元件,而且包含多种胁迫诱导元件,特别是在序列中发现一些苯丙氨酸解氨酶特有的元件。为了鉴定JcPAL基因的启动子元件,分别将长度不同的5′端侧翼区缺失体定向插入载体pBI121中,取代原有的CaMV35S启动子,构建了4个驱动报告基因GUS的植物表达载体。

Phenylalanine ammonia-lyase （PAL） plays a crucial role in phenylpropanoid metabolism and provides precursors of various phenylpropanoid compounds. The 1334 bp 5＇upstream region of the gene encoding phenylalanine ammonia-lyase was isolated from Jatrapha curcas L. by the DNA walking technology. Sequence analysis revealed that the PAL promoter sequence contains not only CAAT and TATA basic modifs that are conserved in the eukaryotic gene promoter, but also various stress related cis-acting dements. Especially, many cis-elements observed in other phenylalanine ammonia lyase promoters are also found in the JcPAL promoter. For further study on the relationship of organization and function of JcPAL promoter, four JcPALP fragments with different deletion regions were inserted into vector pBI121 （with GUS report gene） to replace CaMV35S promoter and might be used to investigate their corresponding expression pattern.