The Screening Of CBLs Protein Which Interact With ZmCIPK8

CBL is a kind of calcineurin B-like proteins that is known to beunique in plants which is discovered in recent years. It plays an important role in thesignal transduction process of plant response to stress by together with the targetprotein CIPK. Previous studies have shown that ZmCIPK8plays an important role inthe maize response to adversity stress. Analyzing the interaction between ZmCIPK8and CBLs is significant for researching the molecular mechanism of ZmCIPK8response to adversity stress. In the present study, bioinformatics, the yeast two-hybrid,RT-PCR and subcellular localization technology were used to screen and analyze theCBLs which interact with ZmCIPK8. Main conclusions are as follows:(1)10CBL sequences were retrieved using the CBL gene sequences from riceand Arabidopsis in NCBI BLAST database. Phylogenetic analysis find that the maizeCBLs are divided into three groups, CBL4,CBL5,CBL8are divided into a group,CBL10is separately as a group, and the others are divided into a group. Sites ofprotein structure analysis find that all ten CBLs containing three EF-hand domains,and the size of each EF-hand type structure is completely consistent. The link area ofEF-hand domains is also like this. Only both end sequences are different. CBL1andCBL9are in the same evolutionary branched, and they have high homology (99%),and the same functional domains. So we suggest that they may have the same targetproteins.(2) In the yeast two-hybrid system, PGBKT7-ZmCIPK8was used as baitplasmid to screen the CBLs proteins which interact with ZmCIPK8from the tenmaize CBL proteins. The results showed that three transformation groups whichcontain PGADT7-CBL1, PGADT7-CBL4and PGADT7-CBL9plasmids havingcolonies growing in the four short of culture. The X-gal experiment also find that theclonies can indicate blue within8h. So we suggest CBL1,CBL4,CBL9can interact with ZmCIPK8. Their gene accession numbers are EU907931, EU973091, EU960527,respectively.(3) In order to further study the interaction between ZmCIPK8and CBL1, CBL4,CBL9, we analysis the expression of these four genes in the maize under droughtstress. The expression of ZmCIPK8gene in the roots and leaves are slightly increasedin30min, then decreased slowly. The expression of CBL1, CBL4, CBL9aresomewhat elevated in the early treatment, and then gradually reduced in the latterperiod. These results provide a basis for the interaction between these proteins inplant.(4) In order to define the subcellular localization of CBL1,CBL4,CBL9gene,three CBL1,CBL4,CBL9fusion expression vectors containing eGFP were constructedinto the PS1300. We used agrobacterium EH105as mediation to infect onionepidermal cells by transient expression technology. Laser confocal microscopescanning found that CBL1, CBL4, CBL9located in the cell membrane.