‘法兰地’草莓LDOX基因的克隆及其过表达与沉默载体的构建

Cloning of the LDOX Gene of 'Falandi' Strawberry and Construction of Its Overexpression and Silencing Vector

花色素苷是草莓果实的主要呈色物质,无色花色素双加氧酶(LDOX)是花色素苷合成途径中的关键酶。为探究LDOX基因在草莓花色素苷合成途径中的功能特性,构建‘法兰地’草莓LDOX基因的过表达与沉默载体。本试验用RT-PCR方法克隆出‘法兰地’草莓LDOX基因的CDS序列(1 152 bp)及干扰片段(468 bp),并成功用植物表达载体pRI101-An-EGFP及干扰载体pK7GWIWG2(Ⅱ)构建了过表达载体及沉默载体,同时对LDOX基因序列进行了生物信息学分析。经农杆菌介导瞬时转化后,可以观察到绿色荧光蛋白,表明过表达载体及沉默载体已成功转染草莓果实。qRCR验证表明,相比对照组果实,LDOX基因过表达的草莓果实基因表达量增加了3.7倍,沉默果实下降了70%,表明LDOX基因过表达与沉默成功。本研究为进一步探究LDOX基因的功能特性提供指导。

Anthocyanin is the main color substance of strawberry fi-uit, and leucoanthocyanidin dioxygenase （LDOX） is a key enzyme in anthocyanin synthesis pathway. In order to explore the functional characteristics of LDOX gene in the anthocyanin synthesis in strawberry, and construct the overexpression and silencing vector of the LDOX gene of‘Falandi＇ strawberry, this study cloned the CDS sequence （1 152 bp） and interference fragment （468 bp） of LDOX gene in ‘Falandi＇ strawberry by RT-PCR, and successfully constructed the overexpression vector and silencing vector by using the plant expression vector pRI101-An-EGFP and interference vector pKTGWIWG2 （ 11 ）. The study also analyzed the bioinformatics of LDOX gene sequences. After Agrobacteriummediated transient transformation, green fluorescent protein could be observed, indicating that the overexpression vector and silencing vector had been successfully transfected into strawberry fruit. The results of qRCR validation showed that the gene expression of strawberries fruits with overexpressed LDOX gene increased by 3.7 times and that with silence LDOX gene decreased by 70% comparing to the control fi-uits, indicating the overexpression and silence of LDOX gene were successful. This study might lay a foundation for further exploring the functional properties ofLDOX gene.