Characterization Of CBLs/CIPKs From Vitis Amurensis And Expression Analysis Of Cold-stress Responsive Pattern

Grape is one of the oldest plants on the earth,the area and yield of viticulture in our country are at the forefront of the world.But the factors of limiting its development effected the productivity and quality of grape severely,such as chilling injury and freezing injury.CBLs/CIPKs signaling network plays a critical role in Calcium-signal transduction pathway in plants cell in response to adversity stress.Although researches on this signaling network have made much progress in Arabidopsis,rice and other model plant species in the recent years,the research about participating in low temperature stress in the grape has not been reported.This research integrated grapevine(Vitis vinifera)genomic approaches of data mining and homologous cloning were used to obtain a total of 8 CBLs and 19 CIPKs gene family members from cold-tolerant Vitis amurensis,comparative analysis Tissue-specific expression and expression pattern in response to cold stress with grapevine Chardonnay(V.vinifera)which sensitive to low temperature,and using the yeast two-hybrid technique to test the interactions of CBLs and CIPKs,the main results are as follows:1.VaCBLs showed significant protein similarity with VvCBLs,is 67.6%-100%.Bioinformatics analysis show that all CBLs proteins from V.amurensis contained four EF-hands conserved domains,V aCBL6 and VaCBL7 proteins contained one transmembrane motif,VaCBL1 and VaCBL4 contained one MGCXXS/T domain.By Tissue-specific expression of CBLs from V.vinifera as control,using qRT-PCR technique analyze Tissue-specific expression of CBLs from V.amurensis were detected CBLs in all tissues of V.amurensis with different expression intensities,and are different from those from V.vinifera.Gene expression profiles of CBLs are different during the time-course of cold stress.CBL1,CBL4,CBL8 positively regulate plants response to-2? low temperature stress,however CBL5,CBL6 negatively regulate plants response to-2? low temperature stress,and a high level of CBL4 expression was detected 1h after-2? cold stress with 2.07-fold than that of unstressed control.2.VaCIPKs showed significant protein similarity with VvCIPKs,is 36.4%-100%,all CIPKs proteins from V.amurensis contained three classical domains.Among VaCIPK1,VaCIPK2 and VaCIPK13 proteins contained one transmembrane motif,VaCIPKs proteins can be divided into four types according to the homology.Moreover,tissue-specific expression profile of CIPKs gene family members and their responses to cold stress were analyzed in both V.amurensis and V.vinifera.The results showed that VaCIPKs and VvCIPKs expressed relatively low expression in shoots.VaCIPKs all overexpression by induce of-2?cold stress,the high levels of VaCIPK16,VaCIPK19 expression were detected 3h after-2 ? cold stress with 402.05-fold and 615.98-fold than that of unstressed control respectively.But VvCIPKs are not sensitive to 2?,0? and-2? cold stress.3.The results of yeast two-hybrid showed that VaCIPK2 can interact with VaCBL1,VaCBL2,VaCB L3,VaCBL4,VaCBL8,and VaCIPK14 can interact with VaCBL2,VaCBL3,VaCBL4,VaCBL8.The results provide reference basis for exploring the possible function and mechanism of the members of those two gene families in response to cold stress,which lays the theoretical for cold-(frozen-)tolerant molecular improvement in grapevine.