与灵芝酸生物合成相关的细胞色素P450基因的筛选与分析  

Identification and Analysis of Candidate Cytochrome P450 Genes Involved in the Biosynthesis of Ganoderic Acid in Ganoderma lingzhi

作　　者：姜露熙 王俊杰 李娜[2] 徐军伟[1] JIANG LuxiI;WANG JunjieI;LI Na;XU JunweiI(Faculty of Life Science and Technology,Kunming University of Science and Technology Kunming,Yunnan 650500,China;Faculty of Science,Kunming University of Science and Technology,Kunming,Yunnan 650500,China)

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》2018年第2期35-41,168,共8页Acta Edulis Fungi

基　　金：国家自然科学基金(21566016和31360495)

摘　　要：首先采用荧光定量PCR的方法通过分析在液体静置和振荡培养条件下基因的表达量对灵芝(Ganoderma lingzhi)细胞中14个与类固醇结合的P450基因进行筛选;其次在不同培养条件下(振荡、静置、静置钙离子添加和静置氮缺陷)通过分析灵芝酸T积累与P450基因表达的相关性对差异表达显著的P450基因进行进一步筛选;最后通过反义RNA技术对所筛选出的基因进行沉默,分析在灵芝细胞内沉默该基因对灵芝酸T含量的影响。结果表明:14个P450基因中,5个基因(cyp512a3、cyp512a11、cyp5144n1、cyp512u4、cyp512v2)的表达量在静置培养条件下显著高于振荡培养条件,差异表达倍数分别为7.2、3.7、3.4、89.8、63.4;cyp512v2基因的表达与灵芝酸T的积累显著相关;三株cyp512v2基因沉默效率较高的菌株中灵芝酸T含量分别比野生型菌株的含量降低13%、69%、66%,表明cyp512v2在灵芝酸T的生物合成过程中具有重要的作用。Fourteen candidate steroid binding P450 genes involved in the biosynthesis of ganoderic acids （GAs） in Ganoderma lingzhi were first screened by comparison of gene relative expression levels in G. lingzhi between liquid static and shaking cultures using the quantitative fluorescence PCR method, then further screened by correlation analysis between expression level and GA-T content under different culture conditions including shaking, static, static with the addition of calcium and static with nitrogen-limiting, and finally confirmed for activity through the antisense RNA technology. The results showed that expression levels of P450 gene cyp512a3, cyp512all, cyp5144nl, cyp512u4 and cyp512v2 under the static culture condition were 7.2, 3. 7, 3.4, 89. 8 and 63. 4 folds, respectively, compared to those under the shaking culture condition. Among the five P450 genes, cyp512a3 and cyp512 v2 showed a positive correlation between gene expression and GA-T content. Compared to the wild G. lingzhi, the GA-T content in three cypS12v2silence G. lingzhi strains decreased by 13%, 69% and 66%, respectively. Our results suggested that P450 cyp512 v2 may play an important role in the biosynthesis of ganoderic acid T in G. lingzhi.

关 键 词：灵芝 灵芝酸 甲羟戊酸途径 细胞色素P450 反义RNA技术 

分 类 号：S567.31[农业科学—中草药栽培]