Function Study Of Cassava CIPK Proteins In Response To Abiotic Stress Research

Cassava is one of the three potato varieties around the world,widely grown in tropical and subtropical area,it is mainly distributed in sounth china region.Cassava is an energy crop that is tolerant of multiple abiotic stresses,it has high economic benefits,Therefore,it has extremely significance research value that explore potential resistant environment stresses mechanism in cassava.CBL is able to identify and binding the calcium signal in plant,CIPK is a kind of serine/threonine proteins kinase,CBL-CIPK signaling pathways plays key role in plant growth and response to abiotic stresses.In this study,we cloned CIPK family genes from cassava genome and analyzed its expression pattern be response to abiotic stresses,the further study of cassava CBL-CIPK signal pathway.The main results are as follows:1.26 MeCIPK genes were identified from the genome database of cassava,and the MeCIPK family genes was named,and the MeCIPK family genes was cloned by polymerase chain reaction(PCR)method.The phylogenetic tree of cassava MeCIPK family proteins was constructed by bioinformatics technology,and the CIPK family genes structures and the cis-acting element of MeCIPK promoter were analyzed.Showed that MeCIPK protein C-terminal control domain contains conservative NAF/FISL and PPI domain structure,MeCIPK8 and AtCIPK8,MeCIPK21 and AtCIPK21,MeCIPK24 and AtCIPK24 in the sequence is very similar,speculated that they might have similar functionality.MeCIPK family gene promoter regions containing potential cis-acting regulatory elements response to abiotic stresses:cis-acting element involved in heat stress responsiveness(HSE),cis-acting element involved in low-temperature responsiveness(LTR),MYB binding site involved in drought-inducibility(MBS),cis-acting element involved in the stress responsiveness(TC-rich repeats).2.According to the result of RT-PCR,suggest that MeCIPK family genes transcript levels difference in test tissues and organs in the whole life cycle of cassava.MeCIPK5,6,9,10 were constitutively expressed in alltissue and all development stages,whereas MeCIPK16,19,20 are opposite,MeCIPK16,20 is mainly expressed in flowers.By quantitative real-time PCR analysis of MeCIPK family genes in 200 mM NaCl,20%PEG 6000,heat and cold conditions,MeCIPK family genes level of expression will up-regulated when cassva subject to abiotic stresses.MeCIPK4,5,11,16,20,22,24 in roots and MeCIPK14,20 in leaves were up-regulated at the 3 h by salt stress treatment.In 9 h PEG-treated,MeCIPK11,17,19,25 were up-regulated in root.However,only MeCIPK11 was induced in leaves after PEG treatment.MeCIPK7 was significantly induced in roots under cold condition,MeCIPK10,13 in roots and MeCIPK12,16 in leaves were also affected under cold condition.The expressin of MeCIPK19 in roots and MeCIPK2,4,17,25 in leaves was induced under heat condition at both time-points.Therefore,it can be speculated that the family genes responds to abiotic stresses,indicating that the cassava CIPK family genes may play key roles in response to abiotic stresses.3.MeCIPK proteins fusion with green fluorescent protein GFP,used confocal microscope examine transmit into fusion protein of Arabidopsis protoplast where them location,the results found that 12 MeCIPKs protein scatter throughout the cell,but the main locate in the cytoplasm.4.Constituting yeast two-hybrid expression vector of MeCIPK genes,the interrelationship between MeCIPK protein and cassava MeCBL protein was studied by yeast two-hybrid system,and 83 pairs of interaction proteins were found.As for MeCIPK24,an ortholog of Arabidopsis AtCIPK24(AtSOS2),it could strongly interact with MeCBL2,6,10 which suggests that MeCIPK24-MeCBL2,MeCIPK24-MeCBL6 and MeCIPK24-MeCBL 10 might play a role participate in process of be respond to salt stress in cassava.According to the result of the MeCIPK interaction with MeCBL and the expression analysis of MeCIPK family genes responses to abiotic stresses,we preliminary reveal that potential CBL—CIPK signaling networks in cassava response to abiotic stresses,in order to provide guidance for the research of cassava resistance,establish pathway for cassava genetic improvement.