核盘菌转录因子SsMCM1原核表达与亚细胞定位

Prokaryotic expression and subcellular localization of transcription factor SsMCM1 in Sclerotinia sclerotiorum

核盘菌[Sclerotinia sclerotiorum (Lib.) de Bary]是一类寄主范围广泛的植物病原真菌。MADS-box蛋白家族基因广泛存在于生物体中,参与调控细胞识别、新陈代谢、细胞周期等。核盘菌转录因子SsMCM1调控其子实体形成与致病性。为进一步揭示SsMCM1的作用机制,本研究以核盘菌野生型菌株uf-70的cDNA为模板,扩增得到SsMCM1基因,并与原核表达载体pET-28a(+)连接,构建成重组质粒转化到大肠杆菌BL21(pLysS)表达菌株中,通过IPTG的诱导和亲和层析,纯化得到SsMCM1蛋白,并以此为抗原获得抗血清,完成效价测定。利用特异性抗血清,与核盘菌不同组织蛋白进行特异性的免疫结合,发现其与核盘菌总蛋白、核盘菌细胞质总蛋白均特异性结合,而核盘菌细胞壁蛋白未发现特异性反应,表明SsMCM1不定位于核盘菌细胞壁上。此外,构建其亚细胞定位载体pCG-1301::SsMCM1,并将重组质粒转化到农杆菌中,通过农杆菌的侵染作用进行瞬时表达,发现SsMCM1定位于细胞核中,作为转录因子调控核盘菌的子实体发育与致病性。

Sclerotinia sclerotiorum ( Lib.) de Bary is a typical filamentous homothallic fungus which is a serious plant pathogenic fungi and has a wide range of hosts. MADS-box protein family genes are widely present in a variety of organisms and participate in the regulation of cellular functions,including cell recognition,metabolism and cell cycle. The transcription factor SsMCM1 is involved in the fruiting body formation and virulence. To clarify the role of SsMCM1 in S. sclerotiorum mycelial growth and pathogenicity,this study considered the wild-type strain uf-70 cDNA as a model and obtained SsMCM1 gene through PCR specific amplification. The resulting gene fragment was ligated into the prokaryotic expression vector pET-28a ( +) and the recombinant plasmid was transformed into E. coli BL21 ( pLysS) expression strain. The protein SsMCM1 was purified after IPTG induction and affinity chromatography and used as antigen by using the combination of muscle subcutaneous immunization to obtain the appropriate antiserum and complete potency. The specific antiserum was found to have a specific combination with total protein and cytoplasmic protein in S. sclerotiorum. However,no specific response was found in cell wall proteins. It is speculated that the protein is not located in S. sclerotiorum cell wall. On the other hand,by constructing the subcellular localization vectors pCG-1301 and expressing it transiently via the Agrobacterium infusion. The result shows that SsMCM1 is located in the nucleus,which acts as a transcription factor to regulate the fruiting body and pathogenicity in S. sclerotiorum.