| Environmental stresses, such as low temperature, drought and salinity, play an essential role in the development process of plants. In response to these stress, plants establish a set of self-protection to response stress signals in the process of long-term evolution. To fight against kinds of biotic and abiotic stress, the important defense system is mediated by intracellular second messenger-Ca2+ signal of the signal transmission mechanism. During the process, CBL (calcium neural similar protein phosphatase B, Calcineurin B-like proteins), an important binding protein, interactes with the downstream protein CIPK (calmodulin nerve phosphatase similar protein interactions protein kinase B, CBL-interacting protein kinase), and form the CBL-CIPK signal system in the plant.At present, CBL-CIPK system studies mainly concentrated on Arabidopsis and Rice, as well as other plants, however little is know about this system in sugarcane. Therefore, the identification and functional characterization of CBLs and CIPKs genes in sugacane are of vital significance to achieve the goal of sugarcane breeding and also supplement to the elaboration of mechanism of sugarcane resisting stress. In the present study, firstly, the ESTs or Unigene sequences related to sugarcane ScCBLs and ScCIPKs genes were obtained from the NCBI databases and sugarcane transcriptome databases from our lab; secondly, in silico doing combined with RT-PCR (Reverse transcription-PCR) method was used to get the full-length cDNA sequence of four ScCBLs and ten ScCIPKs genes, and the corresponding bioinformatics analysis was also conducted; thirdly, real-time fluorescent quantitative PCR (RT-qPCR) was applied to analyse the tissue-specific expression and also the expression characteristics of these genes under variouis exogenous stresses; finally, Bimolecular Fluorescence Complementation (BiFC) technology was used to explore the protein interaction between ScCBL2 and ScCIPK15. The main results were as follows:(1) Through electronic cloning and homology cloning, four ScCBLs and ten ScCIPKs family genes were cloned. Then, we use the sequences to forecast primary and secondary structure of encoded proteins on line, and predict the function and distribution of these proteins. They belong to ScCBL and ScCIPK family genes, and they are highly similar to sorghum and rice protein homologous amino acid sequence and structure of the CBL and CIPK proteins. These CBL proteins contain EF-hands design domain, Ca2+ binding sites and EFh superfamily structure domain, while CIPK proteins contain PKc-like superfamily and AMPKA-C-like superfamily, a CBL interaction binding sites and a potential PP2C binding sites. Evolutionary analysis results show that the sugarcane CBL and CIPK proteins and those from other plant species can be classified into their respective subtribe.(2) RT-qPCR analysis showed that ScCBLs and ScCIPKs gene families expressed in bud, stem pith, leaf, meristerm and stem skin of sugarcan. Four ScCBLs genes have higer expression in meristerm, bud and stem pith. As for ten ScCIPKs gene:ScCIPK4, ScCIPK15 and ScCIPK21 have higher expression in the stem skin; ScCIPKS, ScCIPK1O, ScCIPK14 have higher expression in bud; only ScCIPK8 has higher expression in meristerm; ScCIPK9, ScCIPK17, ScCIPK28 expression is higher in leaf.(3) RT-qPCR analysis also showed that particular ScCBLs and ScCIPKs of sugarcane have different response models under various stress treatments to abiotic exogenous stresses, such as ABA, NaCl, PEG and CaCh, and the same stess can even be sensed by different members of ScCBLs and ScCIPKs family genes. The above results shows that different sugarcane ScCBLs and ScCIPKs genes play a common role in the response to biotic and abiotic stresses. In addition to the biological stress-Mosaic Virus stress, these target genes also show different expression levels under various stresses.(4) The interaction between sugarcane ScCBL2 and ScCIPKl 5 proteins was confirmed by using Bimolecular Fluorescence Complementation technique analysis. This suggests that it does exist interaction through the formation of CBL-CIPK compounds to decode the specificity of Ca2+ signal to transduct signal stress in sugarcane. |
