山葡萄低温诱导酵母双杂三框cDNA文库构建和VaCIPK18互作蛋白筛选鉴定

类钙调磷酸酶B亚基蛋白CBL(calcineurinB-likeprotein)与其互作蛋白激酶CIPK(CBL-interacting protein kinase)组成的CBL-CIPK系统,在真核生物钙信号转导及各种胁迫应答途径中发挥重要作用。采用SMART与LD-PCR技术构建了山葡萄‘双丰’(Vitis amurensis Rupr.‘Shuangfeng’)叶片组织低温胁迫下的均一化酵母双杂交三框cDNA文库,库容量分别为1.7×106、1.3×10⁶与1.9×10⁶cfu·mL(-1),外源基因插入片段长度约为500～2 000 bp,重组率为100%,符合后续双杂交筛选要求。以本课题组前期鉴定受低温诱导表达、具有核质亚细胞定位的山葡萄VaCIPK18全长(VaCIPK18)、激酶结构域缺失(VaCIPK18^△S_TKc)以及NAF结构域缺失(VaCIPK18^△NAF)为诱饵,进行酵母双杂交筛选,获得了17个候选互作靶蛋白;从中选取并克隆了7个参与非生物胁迫的候选互...

Construction of Yeast Two-hybrid Three-frame cDNA Library of Vitis amurensis and Screening of VaCIPK18 Interaction Protein

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.