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

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

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,经木薯再生体系获得转基因苗,在添加抗生素的培养基上进行生根筛选,经PCR鉴定获得Mechlppdk的干扰株系。用qPCR的方法鉴定Mechlppdk在干扰株系的表达情况。结果表明,Mechlppdk在木薯叶片中表达量最高,达到管家基因的50%。在Mechlppdk的转运肽区,选取1个保守区段设计引物,构建RNA干扰表达载体pART27-Mechlppdki,将表达载体转化农杆菌LBA4404,侵染木薯脆性胚愈伤,获得阳性转基因木薯苗7个株系。通过qPCR分析发现各株系Mechlppdk转录本有不同程度的降低。通过以上分析表明本研究获得了干扰效率较高木薯Mechlppdk干扰株系,将为解析木薯Mechlppdk的功能提供研究材料。

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.