冬虫夏草的成熟伴随着冬虫夏草子座中多个基因突变型冬虫夏草菌表达的变化.pdf

北京大学学报(医学版
JOURNAL OF PEKING UNIVERSITY(HEALTH SCIENCES)Vol44 No.3
2012
论著
冬虫夏草的成熟伴随着冬虫夏草子座中多个
基因突变型冬虫夏草菌表达的变化
高凌,李晓红2,赵建晴,陆继红,赵家剛,朱佳石56?
(1.如新华茂公司北京临床药理研究中心,北京10008;:2.博奥生物有限公司,北京102206;3, University of
Califomia, San Diego School of Medicine, La Jolla, CA 92093, USA; 4. Nu Skin Center for Anti-aging Research, Provo
UT84601,USA;5.新疆石河子大学药学院,石河子8320006.香港理工大学应用生物学和化学科技系,香港
[摘要]目的:检测冬虫夏草成熟过程中冬虫夏草菌转换突变和颠换突变基因型在子座中表达的动态变化。方
:采用 MASSARRAY単柲背酸多态性( single nucleotide polymorphism,SNP)质谱基因分型法,根据冬虫夏草茵基因
内转录问隔区( internal transcribed spacer,IS)序列中大量散在的点突变,设计了8个SNP延伸引物,其中5个位于
RDNA ITS1和S2段:067721-211,067721-240,067721-471,067021-531和067721-581,用于检验C和AT偏倚基因
型,但不区分两个AT偏倚基因型;另外3个延伸引物位于rDNA5.8S段:067140-324,067740-328和067740-360,用
于区分两个AT偏倚基因型。应用基质辅助激光解吸附电离飞行时间质谱法( matrix- assisted laser desorption/ Ionize
ion- -time of flight, MALDI-TOF)测定各个SNP位点的单核苷酸延伸反应,观察未成熟和成熟冬虫夏草子座中转换突
变和颠换突变基因型表达的动态变化。結暴:冬虫夏草子座rDNA的8个SNP位点的质谱图显示复杂的动态变化。
067721-211位点的AT偏倚型等位基因的分子浓度明显高于GC偏倚型,但随着冬虫夏草的成熟,这个位点的AT
型等位基因消失。多个SNP位点的质谱图中不仅显示转换突变的等位基因,颠换突变等位基因也有很高的检出
率,有些甚至超过GC和AT偏倚基因型等位基因的峰高;而随着冬虫夏草的成熟,颠换突变等位基因的检出率和
峰高趋于下降。在区分两个AT偏倚基因型的研究中,ABO67744和ABO67740两个基因型的等位基因同时存在于
未成熟冬虫夏草子座中。随着冬虫夏草的成熟,ABO67744基因型等位基因的峰高大幅度降低,而ABO67740基因
型等位基閃的峰高大幅度升高。论:在冬虫夏草子座中共存的至少5个冬虫夏草菌突变基因型的表达随着冬虫
夏草的成熟呈现动态变化,这些突变基因型菌的存在和它们的动态变化对于冬虫夏草子座的萌发和成熟具有重要
意义
[关键词了冬虫夏草;多态性,单核苷酸;突变;成熟
[中图分类号]R282.71[文献标志码]A[文章编号11671-167X(2012)03-0454-10
doi:10.3969/j.issn.1671-167X.2012.03.025
Maturation of Cordyceps sinensis associates with alterations of fungal expressions of
multiple Ophiocordyceps sinensis mutants in stroma of Cordyceps sinensis
GAO Ling, LI Xiao-hong,, ZHAO Jian-qing, LU Ji-hong ZHAO Jia-gang, ZHU Jia-shit*%
(1. Pharmanex Beijing Clinical Pharmacology enter, Beijing 10008, China; 2. Capital Bio Co, Beijing 102206, Chi
na; 3. University of California, San Diego School of Medicine, La Jolla, CA 92093, USA; 4. Nu Skin Center for Anti
Aging Research, Provo, UT 84601, USA; 5. School of Pharmacy, Shihezi University, Shihezi 832000, China
6. Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University, Hong Kong, China)
ABSTRACT Objective: To examine maturational changes in expressions of Ophiocordyceps sinensis(O
sinensis transition and transversion mutation genotypes in Cordyceps sinensis C. sinensis) stroma
Methods: Mass ARRAY single nucleotide polymorphism(NP)matrix-assisted laser desorption/ioniza
tion-time of flight(maldi-tof)mass spectrum genotyping was used, and 8 SNP extension primers were
designed based on the scattered, multiple point mutations of known sequences for the O. sinensis mutants
within their internal transcribed spacer (ITS )segments. Of the extension primers, 5(not capable of dis-
tinguishing between the 2 At-biased genotypes located in RDNA ITS1 and ITS2 regions: 067721-211
067721-240, 067721-477, 067721-531 and 067721-581. The other 3 extension primers located in 5. 8S
RDNA region: 067740-324, 067740-328 and 067740-360, to distinguish between the 2 At-biased geno-
types Results: MS chromatograms at the 8 SNP sites showed dynamic alterations of mutant alleles in C.
基金项目:华茂冬虫夏草研究基金和博奥生物有限公司科研基金资助 Supported by the Pharmanex Fund for Cordyceps Sinensis Research and
the Capital Bio Research Fund
ACorrespondingauthorse-mail,zhujosh@pharman