谷氨酸合酶在灵芝中生物学功能的研究  

Study on the biological function of glutamate synthase in Ganoderma lucidum

作　　者：朱静 岳思宁 陈琛 宋书琪 连玲丹 赵明文[1] ZHU Jing;YUE Sining;CHEN Chen;SONG Shuqi;LIAN Lingdan;ZHAO Mingwen(College of Life Sciences/Key Laboratory of Edible Mushroom Processing,Ministry of Agriculture and Rural Affairs,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学生命科学学院/农业农村部食用菌加工重点实验室

出　　处：《南京农业大学学报》2019年第6期1073-1079,共7页Journal of Nanjing Agricultural University

基　　金：中国博士后科学基金项目(2017M611835);江苏省自然科学基金项目(BK20180535)

摘　　要：[目的]通过对谷氨酸合酶(glutamate synthase,GOGAT)的基因序列进行预测和分析,构建谷氨酸合酶基因的沉默转化子,验证谷氨酸合酶在灵芝吸收、利用氮源、生长及次级代谢中的作用。[方法]利用真菌中已报道的谷氨酸合酶基因,在灵芝基因组库中分析比对得到灵芝中可能的谷氨酸合酶基因。通过构建进化树和结构域分析及酶活性测定,确定灵芝谷氨酸合酶序列。随后利用RNA干扰技术,将灵芝的GOGAT基因进行沉默,研究GOGAT对灵芝生长及次级代谢的影响。[结果]获得灵芝谷氨酸合酶序列,该酶具有保守的谷氨酸合酶结构域,并且在进化上高度保守。在4种氮源(谷氨酰胺、铵盐、脯氨酸和硝酸盐)条件下检测谷氨酸合酶活性和基因转录水平,野生菌株中的GOGAT比酶活和转录水平在不同氮源条件下没有显著变化。通过RNA干扰方法,获得沉默效率分别为(77.0±1.9)%和(77.5±2.0)%的GOGAT沉默菌株2株。平板试验表明,将GOGAT沉默后,灵芝菌丝的生长受到一定程度的抑制。在硝酸盐培养条件下,相比于野生菌株,GOGAT沉默菌株基本停止生长;而在脯氨酸存在条件下,2株沉默菌株的生长直径分别降低(16.0±1.9)%和(21.0±1.3)%。进一步检测灵芝三萜含量发现,当使用铵盐或脯氨酸作为唯一氮源培养时,相比于对照菌株,GOGAT沉默菌株中的灵芝三萜含量分别降低(32.1±4.5)%和(11.0±1.1)%;而以谷氨酰胺和硝酸盐培养时,各菌株中的灵芝三萜含量没有显著变化。[结论]本研究获得了灵芝中合成谷氨酸的重要酶基因GOGAT,发现其对灵芝的生长及次级代谢十分重要。[Objectives]By predicting and analyzing the gene sequence of glutamate synthase(GOGAT),GOGAT silenced strains were constructed to verify the roles of GOGAT in nitrogen utilization,growth,metabolism biosynthesis in Ganoderma lucidum. [Methods]Using the sequence of glutamate synthase that had been reported in other fungi,the probable sequence of glutamate synthase was obtained by comparison in the genome library of G. lucidum. The sequence of glutamate synthase was determined by constructing a phylogenetic tree,domain analysis and enzyme activity assay. RNA interference technology was used to construct the GOGAT silenced strains,and the effects of GOGAT on fungal growth and secondary metabolism was studied in G. lucidum. [Results]The sequence of glutamate synthase of G. lucidum was obtained,which had conserved glutamate synthase domains and was highly conserved in evolution. The enzyme activity and transcription level of glutamate synthase gene were analyzed under different nitrogen conditions,and no significant changes were observed. Two GOGAT silenced strains were constructed with a silencing efficiency of(77.0±1.9)% and(77.5±2.0)%,respectively. The mycelium growth was inhibited when GOGAT was silenced. The mycelia of GOGAT silenced strains stopped growth compared with wild type(WT),when using the nitrate as the nitrogen source. While the diameter of the colony decreased(16.0±1.9)% and(21.0±1.3)%,respectively,in the GOGAT silenced strains under the cultivation of proline. The contents of ganoderic acid in the GOGAT-silenced strains decreased about(32.1±4.5)% and(11.0±1.1)%,respectively,when ammonia or proline was used as the sole nitrogen source. But it showed no difference among control strains and GOGAT silenced strains when cultured with glutamine or nitrate. [Conclusions]The sequence of glutamate synthase in G. lucidum was obtained. We found that the GOGAT was important for fungal growth and secondary metabolism in G. lucidum.

关 键 词：灵芝 谷氨酸合酶 基因沉默 氮源 菌丝生长 灵芝三萜合成 

分 类 号：Q936[生物学—微生物学]