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

doi:10.11924/j.issn.1000-6850.casb2020-0359

Abstract

Abstract:

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.

Key words: pyruvate phosphate dikinase, expression profile, RNA interference, expression vector, Manihot esculenta, genetic transformation, friable callus, transgenic plantlets, PCR identification

CLC Number:

S533

Wang Haiyan, Chen Xin, Zhou Xincheng, Shen Xu, Kong Hua, Wang Wenquan. Expression Analysis of Mechlppdk Gene in Cassava and Construction of RNA Interference Vector and Genetic Transformation[J]. Chinese Agricultural Science Bulletin, 2021, 37(17): 19-25.

Figures/Tables 8

References 28

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