梯棱羊肚菌类膨大素蛋白Cerato-Platanin(CP)生物信息学分析及原核表达

Bioinformatics Analysis and Prokaryotic Expression of Morchella importuna Cerato-Platanin Protein

梯棱羊肚菌Morchella importuna是一种珍稀食药用菌,经济价值极高,但其生活史及子实体发育过程并未完全明确,栽培技术尚不稳定。通过查阅文献和比对梯棱羊肚菌基因组信息,找到2个Ceratoplatanin(CP,类膨大素)家族的基因,分别命名为Mi-CP1与Mi-CP2。采用生物信息学方法分析了Mi-CP1和Mi-CP2蛋白的结构及理化性质,构建了系统发育树,并采用qRT-PCR检测Mi-CP1和Mi-CP2基因在菌丝期、白菌核期、黄菌核期、原基期、幼菇期、成熟子囊果期6个不同生长发育阶段的相对表达量。结果显示:Mi-CP1仅在菌丝期显著表达,而Mi-CP2在原基期和幼菇期大量表达,其中原基期是幼菇期的4.7倍,表明Cerato-platanin(CP)蛋白不仅与梯棱羊肚菌的营养生长有关,还可能参与了子囊果的发育。通过去除信号肽,分别构建pMAL-c2X-CP1X和pMAL-c2X-CP2X的原核表达载体,在大肠杆菌BL21(DE3)菌株中诱导表达,结果仅Mi-CP1X蛋白成功诱导表达,而Mi-CP2X蛋白未能成功诱导表达,推测可能与使用的表达载体有关。本研究为揭示CP蛋白在羊肚菌生长发育过程中的作用奠定了基础。

Morchella importunais a kind of rare edible and medicinal fungus with high economic value,however,the life history of M.importuna and its fruit body development process are not completely clear,resulting in its unstable cultivation.In this paper,two Cerato-platanin(CP,Expansin-like)family genes were found from the literature and comparing the genome of M.importuna.And the structure and physicochemical properties of Mi-CP1 and Mi-CP2 proteins were analyzed by bioinformatics,a phylogenetic tree was constructed,and the relative expression levels of Mi-CP1 and Mi-CP2 were measured through qRT-PCR analysis in six different growth and development stages of mycelium,white sclerotium,yellow sclerotium,primordium,young fruitbody,and mature ascocarp stage.The results showed that Mi-CP1 was only significantly expressed in the hyphae stage,while Mi-CP2 was highly expressed in primordial and young mushroom stage,and the expression in primordial stage was 4.7 times higher than that in young mushroom stage,indicating that Cerato-platanin(CP)protein was not only related to the vegetative growth of M.importuna,but also involved in the development of ascocarp.By removing the signal peptide,the prokaryotic expression vectors of pMAL-c2 X-CP1 X and pMAL-c2 X-CP2 X were constructed and induced to express in E.coli BL21(DE3)strain,respectively.The results showed that only Mi-CP1 X protein was successfully expressed instead of Mi-CP2 X protein probably owing to the expression vector.This study laid a foundation for revealing the role of CP protein in the growth and development of M.importuna.