灵芝细胞中异源表达透明颤菌血红蛋白基因提高胞外多糖的产量  

Heterologous Expression of Vitreoscilla Hemoglobin in Ganoderma lingzhi for Increased Exopolysaccharide Production

作　　者：刘柯 李焕军[1] 张德怀 岳同辉 李娜[2] 徐军伟[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500 [2]昆明理工大学理学院,云南昆明650500

出　　处：《食用菌学报》2017年第3期35-41,共7页Acta Edulis Fungi

基　　金：国家自然科学基金项目(31360495和21566016)资助

摘　　要：在灵芝(Ganoderma lingzhi)中异源表达透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)基因,采用一氧化碳差异波谱分析的方法检测转基因菌株的生物活性,并对野生型菌株和工程菌株进行发酵,通过荧光定量PCR对灵芝多糖生物合成途径上的葡萄糖磷酸变位酶(α-phosphoglucomutase,PGM)、尿苷二磷酸葡萄糖焦磷酸化酶(uridinediphosphate glucose pyrophosphorylase,UGP)和β-1,3-葡聚糖合酶(β-1,3-glucan synthase,GLS)3个基因的转录水平进行分析。研究表明:透明颤菌血红蛋白基因(Vitreoscilla hemoglobin gene,vgb)在灵芝中能够成功表达,并且具有生物活性;工程菌株中胞外多糖的最高产量达0.83g/L,比野生型菌株提高了88.6%;与野生型菌株相比,工程菌株中多糖生物合成途径上PGM、UGP和GLS基因的相对表达量分别是1.52、1.55和3.85。异源表达透明颤菌血红蛋白基因是提高灵芝胞外多糖产量的一种有效方法。In order to increase exopolysaccharide（EPS）production in Ganodermalingzhi,a hemoglobin gene from Vitreoscilla（vgb）was transformed and expressed in G.lingzhi wild-type cells.The bioactivity of vgb translation product VHb protein in transgenic G.lingzhi was confirmed by carbon monoxide difference spectrum analysis.Using quantitative fluorescent PCR,transcription levels of genes involved in polysaccharide biosynthesis,includingα-phosphoglucomutase（PGM）,uridinediphosphate glucose pyrophosphorylase（UGP）andβ-1,3-glucan synthase（GLS）,were measured for both transgenic G.lingzhi and wild-type cell cultures.Compared to wild-type cells,the relative transcription levels of PGM,UGP,and GLS in transgenic G.lingzhi cells were 1.52,1.55 and 3.85,respectively.In line with the enhanced transcription level of polysaccharide biosynthesis pathway,the highest EPS yield in transgenic G.lingzhi cells reached 0.83 g/L,which was 88.6% greater than that in wild-type cells.

关 键 词：灵芝 葡萄糖磷酸变位酶 尿苷二磷酸葡萄糖焦磷酸化酶 

分 类 号：S567.31[农业科学—中草药栽培]