Functional Analysis Of Two Key Stress-related Genes In CBL-CIPKs Regulatory Network In Vitis Amurensis

Grape(Vitis L.)is one of the most important economic fruit trees worldwide.Currently,the widely-cultivated Vitis species are mainly European grape varities,and their quality are excellent,but relatively poor in tolerant traits.Chilling(Freezing)damage is one of the important environmental factors that limit the growth and development of grapevine,which seriously affects the yield and quality of grapevine.Thus,it has become an important problem to be urgently solved in the cool area of northern China.Vitis amurensis is one of the stongest cold-tolerant species in the genus Vitis,which has been widely utilized as an important germplasm resource for grapevine cold-tolerant breedings in the world.The CBL-CIPK regulatory network mediated by Ca2+,plays a significant role in response to various stresses.The functons of some members of this regulatory network have been reported in the model plants Arabidopsis thaliana and rice,however,the related members of CBLs and CIPKs in cold-tolerant V amurensis involved in stress response,are relatively poor.In this study,some key members of CBL-CIPK regulatory network were cloned from this cold-tolerant Vitis species.Moreover,the expression patterns,location,interaction proteins of VaCBL01 and VaCIPK02 genes,were further investigated to explore their functional roles in response to stress response.The main results are as follows:1.The expression characteristics of VaCBL01 and protein interaction analysis with VaCIPKs.VaCBL01 in constitutively expressed in roots,shoots,tendrils,flowers and young leaves of V amurensis,among which a highly expressed level was detected in tendril.VaCBL01 was induced by ABA,and a highly expressed level was at 12 h,followed by a down-regulated expression.Under low temperature(4?)stresss,VaCBL01 displayed a down-regulated tendency.The interaction intensity and specificity were different among VaCBL01 with 19 VaCIPKs,respectively.VaCBL01 strongly interacted with 12 VaCIPKs,and weakly interacted the other five VaCIPKs were weak,but did not interact 2 VaCIPKs.These results indicated VaCBL01 plays an important role in decoding Ca2+ signal and transducing signals after forming CBL01-CIPKs complexes.2.The expression characteristics,subcellular localization,transcription activity,and interacting protein,of VaCIPK02.VaCIPK02 was constitutively expressed in different tissues of V.amurensis,but the expression level was different,among which the expression level in roots were lower and the expression level in tendril were relatively high.VaCIPK02 was up-regulated by 4?,and reached a higher level at 6 h.VaCIPK02 was down-regulated by ABA.The VaCIPK02 protein was located in the nucleus,chloroplast and cell membrane.VaCIPK02 and VaCIPK02?NAF had no self-activating activity,but VaCIPK02?S_TKc had self-activating activity.VaCIPK02 interacts with four CBLs of V.amurensis(VaCBL01,VaCBL04,VaCBL05 and VaCBL08),and VaCIPK02 itself cannot form a homodimer.The yeast cDNA library constructed from leaves of V amurensis induced by low temperature at-2? was screened and 20 interacting proteins were screened.The random hybridization showed that VaCIPK02 protein interacted with VaPYL9.3.The functional analysis of VaCIPK02 gene and its promoter in Arabidopsis thaliana.The promoter sequence of 2,269 bp was homologously cloned,and the ProVaCIPK02::GUS fusion expression vector was constructed to transform A.thaliana to obtain transgenic homozygote.The results of GUS histochemical staining showed that the VaCIPK02 promoter was induced by stress(NaCl,mannitol,4? low temperature)and hormone(ABA)with different intensities.The VaCIPK02-overexpressing A.thaliana plants was obtained by constructing the 35S::VaCIPK02::EGFP plant expression vector and Agrobacterium-mediated transformation.Under the ABA treatment,VaCIPK02-OE Arabidopsis plants showed hypersensitivity compared with WT.