至少6个突变基因型冬虫夏草菌在冬虫夏草子座中表达的动态变化

本研究检测了在冬虫夏草成熟过程中突变基因型冬虫夏草菌在子座中表达的动态变化。根据冬虫夏草菌基因内转录间隔区(ITS)序列中大量散在的点突变,设计了8个单核苷酸多态性(SNP)延伸引物,采用SNP质谱基因分型法测定各个SNP位点的单核苷酸延伸反应,观察冬虫夏草子座中转换突变和颠换突变基因型菌在冬虫夏草成熟过程中表达的动态变化。其中5个SNP延伸引物(067721-211,067721-240,067721-477,067721-531和067721-581)位于rDNA ITS1和ITS2段,用于区分GC和AT偏倚基因型,但不区分2个AT偏倚基因型。另外3个延伸引物(067740-324,067740-328和067740-360)位于rDNA 5.8S段,用于区分2个AT基因型。采用PCR+EcoRⅠ限制性酶切法检验2个GC偏倚基因型冬虫夏草菌在冬虫夏草成熟过程中表达的动态变化。结果表明:冬虫夏草子座rDNA ITS1-5.8S-ITS2片段的8个SNP位点的质谱图谱显示冬虫夏草子座中存在至少5个冬虫夏草菌转换突变和颠换突变等位基因。它们的表达在冬虫夏草成熟过程中呈现动态变化。067721-211和067721-477位点的AT偏倚型等位基因的峰高明显高于GC偏倚型;随着冬虫夏草的成熟,这2个位点的AT型等位基因峰高大幅度下降。SNP质谱图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,它们的峰高在一些位点甚至超过GC和AT基因型等位基因;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2个AT偏倚基因型的研究中,AB067744和AB067740 2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,AB067744基因型等位基因的峰高明显降低,而AB067740基因型等位基因的峰高明显升高。PCR产物EcoRⅠ酶切试验发现子座中存在2组GC偏倚型菌,具有完全不同的成熟模式。综上,冬虫夏草子座中存在至少6个突变基因型冬虫夏草菌,其表达随着冬虫夏草的成熟呈现动态变化。这些突变基因型菌表达的动态变化对于冬虫夏草子座的萌发和成熟具有重要菌物学意义。

Dynamically Altered Expressions of at Least 6 Ophiocordyceps sinensis Mutants in the Stroma of Cordyceps sinensis

The dynamic changes in expressions of Ophiocordyceps sinensis(Os) mutant genotypes in the Cordyceps sinensis(Cs) stroma was examined during Cs maturation.MassARRAY single nucleotide polymorphism(SNP) MALDI-TOF mass spectrum genotyping was used,and 8 SNP extension primers were designed based on the scattered,multiple point mutations of known Os mutant sequences within their internal transcribed spacer(ITS) segments,to observe the maturational changes in expressions of Os mutant genotypes in Cs stroma.Of the extension primers,5 locate in rDNA ITS1/2 regions: 067721-211,067721-240,067721-477,067721-531 and 067721-581,for distinguishing between the GC-and AT-biased genotypes(incapable of distinguishing between the 2 AT-biased genotypes);other 3 in 5.8S rDNA region: 067740-324,067740-328 and 067740-360,for distinguishing between the 2 AT-biased genotypes.PCR amplification and EcoRⅠ restrictive digestion were used to examine the maturation patterns of 2 GC biased genotypes.The MS chromatograms at the 8 SNP sites showed nonparallel,dynamic maturational alterations of mutant alleles in Cs stroma.The peak heights of the alleles for the AT-biased genotypes were higher at 067721-211 and 067721-477 sites than its GC-biased counterparts in premature Cs stroma,but attenuated largely with Cs maturation.Chromatograms displayed not only transition mutation alleles,but also transversion mutants.Compared to those for GC-and AT-biases,some of the transversion mutant alleles displayed higher peaks,but their detection rate and peak heights appeared to be attenuated with Cs maturation.When distinguished between the 2 AT-biases,AB067744 and AB067740 genotype alleles co-existed in premature Cs stroma.The allele peak height for AB067744 genotype was attenuated greatly with Cs maturation,while that for AB067740 genotype elevated.By using PCR amplification and EcoRⅠ digestion,2 GC-biased genotypes were found having completely different maturation patterns.Conclusion: At least 6 mutant genotypes of Os co-exist in Cs stroma.Their expressions are altered dynamically with Cs maturation,and are of extreme mycological importance in Cs stroma germination and maturation.