Screening and Identification of Interaction Proteins of MeAHL19 in Cassava (Manihot esculenta)
ZHANG Jian-Yu1,2, WANG Xiao-Tong1,2, GENG Sha2,3, WU Zhi-Hao2,3, REN Si-Yang1,2, LI Rui-Mei2, YAO Yuan2, GUO Jian-Chun2, LIU Jiao2,*, HU Xin-Wen1,*
1 School of Life Sciences, Hainan University, Haikou 570228, China; 2 Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences/Hainan Institute for Tropical Agricultural, Haikou 571101, China; 3 College of Tropical Crops, Hainan University, Haikou 570228, China
Abstract：The AT-hook motif nuclear localized protein (AHL) family genes contain a unique combination of 1 or 2 AT-hook domains and regulate various biological processes in plants. To explore the biological function of cassava (Manihot esculenta) AHL family gene MeAHL19 and its role in the protein interaction network, in this study, the bait vector pGBKT7-MeAHL19 was constructed using T4 ligase and used to screen the cassava cDNA library via the yeast (Saccharomyces cerevisiae) two-hybrid (Y2H) library screening system. Real-time quantitative PCR method was used to detect gene expression patterns of MeAHL19 and the interacting proteins. The results showed that 20 positive clones were obtained through Y2H screening. Eight candidate interacting proteins were identified by PCR amplification and sequencing alignment. Y2H test confirmed the interaction between MeAHL19 and ribulose bisphosphate carboxylase small chain (RBCS) protein in vitro. BiFC results further confirmed that MeAHL19 interacted with RBCS in Nicotiana benthamiana mesophyll cells. Analysis of gene expression patterns showed that MeAHL19 was mainly expressed in roots, whereas RBCS was expressed at about the same level in roots, stems and leaves; MeAHL19 and RBCS responded to abiotic stress and exogenous hormone responses in cassava. This study provides a reference for further investigation on the biological function of MeAHL19 in stress responses of cassava.
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