以酵母菌雙雜交法系統分析水稻結鈣激活酵素與其結合蛋白之交互作用

Abstract

OSCK1與OSCK2為水稻中於花粉成熟期大量表現的CDPK (calcium-dependent calmodulin-independent protein kinase)基因，由於鈣離子是花粉萌發的必要因素，故預期OSCK基因可能與調控花粉萌發，甚或是先前花粉發育的過程相關。為了探討此二個CDPK的作用機制，先前實驗室利用酵母菌雙雜交法，篩選出五個可能為OSCK1的結合蛋白或下游受質蛋白，命名為OIPs (OSCK1-interacting proteins)，分別為OIP1、OIP13、OIP18、OIP28及OIP30。其中OIP18與OIP28為兩個不同的CDPK，另稱之為OSCK4與OSCK3。由於藉由酵母菌雙雜交法所選殖出的基因多非全長，因此本實驗首先探討當雙方蛋白均為全長時，是否仍具有交互作用，並進而檢視其生理意義。依據水稻基因組資料庫的資訊，設計引子進行RT-PCR，分別獲得OIP13、OIP18及OIP28的全長cDNA；而OIP1及OIP30則因資料庫中查無對應序列，故利用5- RACE獲得全長cDNA。由RT-PCR實驗結果可知OIP18 (OSCK4)與OIP28 (OSCK3)亦於花粉大量表現，故花粉中至少有四種CDPK基因表現，其蛋白產物彼此間是否可形成複合體值得探討，故構築一系列質體分別可表現四種CDPK全長蛋白，或僅激活酵素區塊，一一配對進行酵母菌雙雜交測試，初步結果顯示僅OSCK2-F可與自我或OSCK4-F蛋白產生交互作用，暗示少數特定之CDPK蛋白間有可能以複合體方式存在體內。此外，OIP1、13、18與28全長蛋白均無法與OSCK1-F產生交互作用，暗示其可能並非水稻中OSCK1蛋白的結合對象；而OIP30則恰相反，除了與OSCK1-F具交互作用外，與其他的OSCKs (OSCK2-F、3-F、4-F)之交互作用亦極明顯，暗示其具有生物意義。為了深入研究OIP30蛋白，故製備其專一性抗體，以西方點墨法偵測水稻各組織之OIP30蛋白含量，發現其於癒傷組織與花粉中表現量最高，於幼葉與根部則含量偏低，未來將藉由抗體探討OSCK1與OIP30蛋白的結合關係。

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.