Mechlppdk基因在木薯中的表达分析及RNA干扰载体构建和遗传转化

本研究为了明确丙酮酸磷酸双激酶(ppdk)在木薯中的表达谱,创制Mechlppdk的干扰株系,以期为Mechlppdk的功能研究提供材料.本研究以栽培型木薯Arg7为材料,用qPCR的方法分析Mechlppdk在木薯根、茎、叶中的表达谱;采用RNA干扰的方法,构建Mechlppdk的干扰载体,并遗传转化木薯脆性愈伤,经...

Expression Analysis of Mechlppdk Gene in Cassava and Construction of RNA Interference Vector and Genetic Transformation

The aim of this study is to clarify the expression profiles of pyruvate phosphodikinase (ppdk) in cassava and to create an interfering strain of Mechlppdk, so as to provide materials for the functional study of Mechlppdk. The cultivated cassava Arg7 was used as the research material. The expression profiles of Mechlppdk in cassava root, stem and leaf were analyzed by qPCR method; the interference vector of Mechlppdk was constructed by RNA interference method, and then transformed into friable callus of cassava. The transgenic plantlets were obtained by cassava regeneration system, and rooting screening was carried out on the medium supplemented with antibiotics. The interference lines of Mechlppdk were identified by PCR. The expression of Mechlppdk in the interference strains were identified by qPCR. The results showed that the highest expression level of Mechlppdk was found in cassava leaves, reaching 50% of housekeeper gene. In the transit peptide region of Mechlppdk, a conservative region was selected to design primers to construct the RNA interference expression vector pART27-Mechlppdki. The expression vector was transformed into Agrobacterium tumefaciens LBA4404 and used to infect the callus of cassava friable callus, and seven positive transgenic cassava lines were obtained. qPCR showed that the transcripts of Mechlppdk decreased in different degrees. In this study, we have obtained the cassava Mechlppdk interference lines with higher interference efficiency, which could provide research materials for analyzing the function of Mechlppdk.