Analyses of Interactions between OSCKs and OIPs by Yeast Two-hybrid System

• Main Authors: Li-Jing Lin, 林立菁
• Other Authors: Wei-Ming Leu
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/64622374533395059890

碩士 === 國立中興大學 === 生物科技學研究所 === 93 === OSCK1 and OSCK2 are two CDPK (calcium-dependent calmodulin-independent protein kinase) genes predominantly expressed in mature pollen of rice. As Ca2+ is essential for pollen germination, it is conceivable that CDPKs may play critical roles in pollen germination and/or pollen development. To identify protein substrate for OSCK1 in pollen, we have used the kinase domain of OSCK1 (OSCK1-K) as a bait in yeast two-hybrid screen and obtained five OSCK1-interacting proteins (OIPs), namely OIP1, 13, 18, 28 and 30. As OIP18 and 28 are also CDPK genes, they were renamed as OSCK4 and OSCK3, respectively. In this study, full-length OIP cDNAs were obtained either directly by RT-PCR according to sequence information retrieved from GenBank (for OIP13, 18, 28), or by 5-RACE, in silico sequence assembly, followed by RT-PCR (for OIP1 and 30). Interactions of the full-length form of OIPs with OSCK1 were re-examined by yeast two-hybrid analyses to confirm their biological significance. Since OIP18 (OSCK4) and OIP28 (OSCK3) were detected highly expressed in pollen, similar to OSCK1 and OSCK2, it is interesting to investigate whether their encoded OSCK proteins could form complexes with each other. Paired analyses on four CDPKs revealed interactions only within OSCK2-F itself or between OSCK2-F and OSCK4-F, indicating that some specific CDPKs may exist as multimer in vivo. Except for OIP30, other OIPs (OIP1, 13, 18, 28) all failed to show interactions with OSCK1 in their full-length form and were excluded for further analyses. Oppositely, OIP30 exhibited strong interactions not only with OSCK1-F but also with other OSCKs (OSCK2-F, 3-F, 4-F), suggesting a biological significance. Examinations of various rice tissues revealed that OIP30 protein was most abundant in callus and pollen, but barely detected in young leaves or root.