梯棱羊肚菌凝集素MIL的外源表达及生物活性  

Heterologous Expression and Biological Activity of Lectin MIL from Morchella importuna

作　　者：舒芳 刘伟[1] 张倩倩 龚钰华[1] 边银丙[1] SHU Fang;LIU Wei;ZHANG Qianqian;GONG Yuhua;BIAN Yinbing(Institute of Applied Fungi,Huazhong Agricultural University,Wuhan,Hubei 430070,China)

机构地区：[1]华中农业大学应用真菌研究所

出　　处：《食用菌学报》2019年第3期19-25,F0003,共8页Acta Edulis Fungi

基　　金：湖北省科技创新重点项目(2016ABA100)

摘　　要：对梯棱羊肚菌(Morchella importuna)凝集素编码基因MIL及其编码蛋白进行生物信息学分析,并采用qRT-PCR分析MIL基因在梯棱羊肚菌不同生长发育阶段(菌丝期、白色菌核期、黄色菌核期、原基期、幼菇期、成熟子囊果期)的表达量;构建pET28a-MIL表达载体,在大肠杆菌(Escherichia coli)BL21中外源表达获得了凝集素蛋白MIL-His,测定其生物活性。研究表明,梯棱羊肚菌MIL基因序列为426 bp,其中含有一个RicinB lectin保守结构域,与RicinB-like lectin同源性达95%;qRT-PCR定量分析显示,在从菌丝到子囊果的生长发育过程中,梯棱羊肚菌MIL基因在白色菌核阶段的表达量最高,其次为黄色菌核阶段,幼菇、成熟子囊果和原基阶段次之,在菌丝阶段表达量最低,表明MIL基因可能在梯棱羊肚菌菌核形成中起到重要的调控作用;兔红细胞凝血实验结果显示重组蛋白MIL-His凝集兔红细胞的最小浓度为0.32 mg/mL,同时发现MIL-His具有提高小鼠脾脏淋巴细胞增殖率的活性,与对照相比增殖率达35.3%。Lectin protein and its coding gene MIL from Morchella importuna were subjected to bioinformatics analysis.Using qRT-PCR,the expression of M.importuna MIL gene at different growth stages,including the mycelial stage,white sclerotium stage,mature sclerotium stage,primordium stage,young fruit body stage,and mature fruit body stage was analyzed.Then,an expression vector pET28a-MIL was constructed for heterologous expression of the recombinant lectin protein MIL-His in E.coli BL21.The recombinant MIL-His protein was then studied for its biological activities ex vitro and in vitro.The results showed that the M.importuna MIL gene sequence was 426 bp in length and contained a conserved domain of RicinB lectin with 95%homology to RicinB-like lectin.The qRT-PCR results showed that the expression of M.importuna MIL gene was the highest at the white sclerotium stage,followed by the mature sclerotium stage,young fruit body,mature fruit body and primodium stage,and was the lowest at the mycelium stage.This expression result suggested that MIL may play an extremely important regulatory role in sclerotium formation.The ex vitro activity of the recombinant MIL-His was studied based on the rabbit red blood cell hemagglutination experiment,and the results showed a minimum concentration of 0.32 mg/mL recombinant MIL-His to agglutinate rabbit red blood cells.For the in vitro activity,MIL-His increased the proliferation rate of mouse spleen lymphocytes by 35.3%compared with the control.

关 键 词：异源表达 血凝集活性 免疫活性 菌核发育 

分 类 号：S43[农业科学—农业昆虫与害虫防治]