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New insights into the evolution and functional divergence of the CIPK gene family in Saccharum

苏炜华  Yongjuan Ren  Dongjiao Wang  Long Huang  Hui Ling  Yachun Su  Ning Huang  Hanchen Tang  Liping Xu  Youxiong Que  
【摘要】:【Research background】 CBL-interacting protein kinases(CIPKs) are the primary components of calcium sensors and play crucial roles in plant developmental processes, hormone signaling transduction, and in the response to exogenous stresses. Sugarcane is an important sugar crop that contributes up to about 80% of the world sugar production. To date, CIPK genes have not been investigated in sugarcane. 【Materials and methods】 In this study, sequence and evolution analysis of the SsCIPK genes were conducted using the available sugarcane genome data. In addition, the expression patterns of the CIPK gene family in the presence of abscisic acid(ABA), polyethylene glycol(PEG), and sodium chloride(NaCl) were detected by qRT-PCR. The allogenic expression of ScCIPKs and interactions between ScCIPKs and ScCBLs were also explored. Under salinity stress, the growth status of E. coli cells expressing ScCIPKs was analyzed. Furthermore, their subcellular localization, a bimolecular fluorescence complementation assay(BiFC), and transient overexpression in Nicotiana benthamiana were also investigated.【Result and analysis】 In this study, 48 CIPK genes(Ss CIPKs) were identified from the genome of Saccharum spontaneum. Phylogenetic reconstruction suggested that the SsCIPK gene family may have undergone six gene duplication events from the last common ancestor(LCA) of Ss CIPKs. The gene structure of Ss CIPKs also incurred substantial variations during evolution, with the number of introns varying from 0 to 15. Whole-genome duplications(WGDs) and signal-gene replications served as the driving force for the amplification of SsCIPKs. Ten Sc CIPK genes were amplified from sugarcane(Saccharum spp. hybrids). Expression analyses of ScCIPK genes using qRT-PCR revealed that ScCIPKs are differentially expressed in response to ABA, PEG, and NaCl. Subcellular localization of ScCIPKs revealed that the fusion protein of four ScCIPK::GFP were all expressed in the cytoplasm and plasma membrane, except for ScCIPK17::GFP, which has also been observed in the nucleus. Prokaryotic expression implied that the recombinant proteins of ScCIPK3,-15, and-17 could only slightly enhance growth under NaCl stress conditions, but ScCIPK21 did not. Transient N. benthamiana plants overexpressing ScCIPKs demonstrated that the ScCIPK genes are involved in responding to external stressors through the ethylene synthesis pathway as well as to bacterial infections. Preferential interactions between ScCBLs and Sc CIPKs were observed in BiFC experiments. 【Conclusion】 These results improve our understanding of the evolution and functions of the CIPK gene family in sugarcane complexes as well as provide a basis for selecting appropriate genes for in-depth functional studies of CBL-CIPK in sugarcane.

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