关键词: 山葡萄, 低温胁迫, cDNA三框文库, VaCIPK18, VaPYL9, 互作分析

Abstract: The CBL-CIPK signaling system is comprised of calcineurin B-like protein（CBL）and its interacting protein kinases（CIPK），playing critical roles in response to calcium signal transduction and various stress responses in eukaryotes. A yeast two-hybrid（Y2H）three-frame cDNA library with the leaves of Vitis amurensis Rupr.‘Shuangfeng’under low temperature stress was constructed using SMART and LD-PCR techniques. The capacities of three-frame libraries were 1.7 × 106，1.3 × 106 and 1.9 × 106 cfu • mL-1，respectively，with the inserted fragment of 500–2 000 bp in length，and 100% recombination rate，which could meet with the requirements of yeast two-hybrid screening. A low-temperature-induced gene VaCIPK18 that previously identified by our team，which localized at both nucleus and cytoplasm，was used as a“bait”to obtain three constructs containing the full length cDNA，as well as the two truncations of the S_TKc domain（VaCIPK18△S_TKc）and the NAF domain（VaCIPK18△NAF），respectively. The result showed that a total of 17 candidate interacting proteins of VaCIPK18 were screened from the library，from which 7 previously reported candidate proteins that involved in abiotic stresses were selected and cloned for rotated verification in yeast. As a result，one upstream signal regulator of ABA pathway，VaPYL9 was preliminary confirmed to interact VaCIPK18. Furthermore，Y2H rotation verification and bimolecular fluorescence complementation（BiFC）were used to verify the interaction between them. Our result demonstrated that VaPYL9 specifically interacts with VaCIPK18 and VaCIPK18△NAF protein in yeast，and VaPYL9 could also interact with VaCIPK18△S_TKc and VaCIPK18△NAF protein in plant cells. Interestingly，there is no interaction between VaPYL9 and VaCIPK18 in Arabidopsis protoplast.

Key words: Vitis amurensis, cold stress, Y2H three-frame cDNA library, VaCIPK18, VaPYL9, interactive analysis