柿果实CCD1基因超表达和RNAi表达载体的构建

Construction of over-expression and RNAi-expression vector of CCD1 gene in persimmon fruit

采用CTAB法提取小方柿果肉总RNA,根据带有不同酶切位点的引物获得目的基因柿类胡萝卜素裂解双加氧酶基因(DkCCD1),将扩增的目的基因序列与NCBI登录的基因序列进行核苷酸序列和氨基酸序列比对,发现核苷酸序列相似度均超过99%,氨基酸序列相似度均为100%,由此得到可用于后续试验的目的基因。将得到的目的基因与双元表达载体pCAMBIA1301连接,构建pCAMBIA1301-CCD1超表达载体和具有发卡结构的pCAMBIA1301-CCD1-RNAi表达载体,通过冻融法将载体导入农杆菌EHA105。PCR扩增结果表明:所构建的pCAMBIA1301-CCD1超表达载体和pCAMBIA1301-CCD1-RNAi载体已导入农杆菌,可用于后续的遗传转化研究。

Total RNA was extracted from fruit pulp of persimmon‘Xiaofangshi’with CTAB method.The target gene carotenoid cleavage dioxigenase 1 gene(DkCCD1)was amplified by primers with different restriction enzyme sites.The results of nucleotide sequence and amino acid sequence alignments showed that the similarity of the nucleotide sequence was more than 99% and the similarity of the amino acid sequence was 100%,which indicated that the sequence can be used in the following experiment.The target gene was ligated with the binary expression vector pCAMBIA1301.The over-expression vector pCAMBIA1301-CCD1 and the RNAi expression vector pCAMBIA1301-CCD1-RNAi with hairpin structure were successfully constructed,and then transferred to Agrobacterium EHA105 by freeze-thawing method.PCR amplification results showed that the pCAMBIA1301-CCD1 overexpression vector and pCAMBIA1301-CCD1-RNAi vectors were introduced into Agrobacterium,which could be used for subsequent genetic transformation research.