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

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.方法:采用MassARRAY单核苷酸多态性(single nucleotide polymorphism,SNP)质谱基因分型法,根据冬虫夏草菌基因内转录间隔区( internal transcribed spacer,ITS)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ITS2段:067721-211,067721-240,067721 -477,067721-531和067721-581,用于检验GC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rDNA 5.8S段:067740-324,067740-328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/ionization-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.结果: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,AB067744和AB067740两个基因型的等位基因司时存在于未成熟冬虫夏草子座中.随着冬虫夏草的成熟,AB067744基因型等位基因的峰高大幅度降低,而AB067740基因型等位基因的峰高大幅度升高.结论: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.%Objective-.To examine maturational changes in expressions of Ophiocordyceps sinensis (O. sinensis) transition and transversion mutation genotypes in Cordyceps sinensis (C. sinensis) stroma. Methods; MassARRAY single nucleotide polymorphism (SNP) 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 0. sinensis mutants within their internal transcribed spacer (ITS) segments. Of the extension primers, 5 (not capable of distinguishing 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 genotypes. Results; MS chromatograms at the 8 SNP sites showed dynamic alterations of mutant alleles in C. sinensis stroma. The allele for the AT-biased genotypes at 067721-211 site showed higher peak height than its GC-biased counterpart in the premature C. sinensis stroma, but disappeared with C. sinensis maturation. Chromatograms displayed not only the transition mutation alleles, but also Iransversion mutants. Some of the transversion mutation alleles displayed higher peak heights than those for GC- and AT-biased alleles, but their peak heights and detection rates tended to be decreased with C. sinensis maturation. When distinguishing hetween the 2 AT-biases, AB067744 and AB067740 genotype alleles co-existed in the premature C. sinensis stroma. The allele peak height for AB067744 genotype was greatly decreased with C. sinensis maturation, while that for AB067740 genotype increased. Conclusion; Co-existence of at least 5 transition and transversion mutant genotypes of 0. sinensis and the dynamic changes in their expressions in C. sinensis stroma along with C. sinensis maturation may be of extreme importance in C. sinensis stroma germination and maturation, enabling C. sinensis to complete its life cycle.