竹黄及其分离真菌的遗传多样性分析

【作者】 范英；

【导师】 陈双林；

【作者基本信息】 南京师范大学 ， 微生物学， 2011， 硕士

【摘要】 竹黄(Shiraia bambusicola P. Henn.)是我国一种稀有的寄生性药用真菌,具有极大的药用价值,随着竹黄中具有光敏活性的竹红菌素(竹红菌甲素和竹红菌乙素)的发现,竹黄的应用研究日益受到重视。为了了解竹黄地理居群间的遗传分化,采用ISSR分子标记对江苏、安徽和浙江3省的8个居群共107个竹黄样本进行了遗传多样性的分析。本研究首先探讨了Mg2+、dNTPs、Taq DNA聚合酶、引物和模板DNA等5个因素对ISSR-PCR扩增的影响,通过单因子试验和正交设计方法相结合建立并优化了竹黄ISSR-PCR反应体系。采用优化好的体系,对来自江苏、安徽、浙江三省的8个自然居群共107个竹黄样本进行遗传多样性和遗传结构的分析。结果表明,筛选得到的11条引物共检测出241个位点,其中多态性位点240个,占99.59%,居群水平的多态位点百分率平均为64.21%,其中以浙江天目山为最高(74.27%),江苏宜兴为最低(45.23%)。Nei’s基因多样性指数、Shannon多态性信息指数分别为0.3314和0.4996,表明竹黄的遗传多样性较高。居群Nei’s基因分化系数Gst为0.3314,居群内产生了较大的遗传分化。AMOVA分析表明,居群间变异占39.96%,居群内变异占60.04%,遗传变异主要存在于居群内。8个居群间的遗传距离变化范围在0.1115～0.2453之间,平均为0.1766。居群间的基因流Nm=1.0086,说明居群间存在一定的基因交流。UPGMA聚类分析以及主坐标分析结果显示,地理上距离较近的居群多聚在一起,表现出一定的地域性,说明各居群的亲缘关系与地理分布有着密切关系。为了鉴定竹黄子实体的相关菌,本研究从江苏宜兴、安徽广德以及浙江的安吉和丽水采集的竹黄子实体上分离纯化得到了36株真菌,对它们进行ITS-rDNA序列的系统学分析,利用邻接法和最大简约法分别对这36株菌的ITS序列和其它15条来自GenBank的相关序列进行了分析,结果表明这些真菌中29株属于子囊菌,3株属于担子菌,4株属于半知菌。子囊菌中包括粪壳菌目(Sordariales),肉座菌目(Hypocreales)和格孢腔菌目(Pleosporales)以及梨孢假壳科(Apiosporaceae),其中又以格孢腔菌目和梨孢假壳科成员为优势菌群,其次为肉座菌目和粪壳菌目。更多还原

【Abstract】 Shiraia bambusicola P. Henn. is a parasitic medicinal fungus, which has a very high medicinal value. With the discovery of the photosensitive hypocrellin (hypocrellin A and hypocrellin B), S. bambusicola has increasing got recognition in research and application.In order to understand the genetic differentiation of the medicinal fungus Shiraia bambusicola, the genetic diversity of 107 individuals in 8 populations collected from Jiangsu, Anhui and Zhejiang provinces were studied by using inter-simple sequence repeat(ISSR) analysis. In this study, the effect of different concentrations of Mg2+, dNTPs, Taq DNA polymerase, primer and template DNA was analyzed and the optimized ISSR-PCR reaction system of S. bambusicola was established by the test of the single factoe and orthogonal experiments. Based on the optimized ISSR-PCR reaction system, th genetic diversity and genetic structure of 107 individuals from 8 geographic locations were submitted to analysis. The 11 primers employed produced a total of 241 bands, of which 240 bands (PPB=99.59%) were polymorphic. At population level, the percentage of polymorphic bands waried from 45.23% for Yixing of Jiangsu province to 74.27% for Tianmu Mountain of Zhejiang province, with a mean value of 64.21%. Nei’s gene diversity index is 0.3314 and the index of Shannon’s genetic diversity was 0.4996, which indicates high level of genetic variability. Genetic differentiation mainly occurred within population (Gst=0.3314). AMOVA detected that variation among populations was 39.96%, variation within populations was 60.04%. Nei’s genetic distance between populations ranged from 0.1115 to 0.2453, with a mean value of 0.1766. Gene flow between 8 populations was 1.0086, indicating a little genetic intercourse. The results of unweighted pair group method arithmetic average (UPGMA) analysis and Principal coordinates analysis (PCA) had reveled that the populations with near distance in geography got together frequently and showed regional characteristic. It showed that the relations between sibship and geographical distribution were intensive.In order to identify the related fungi associated with fruiting body of S. bambusicola collected from Yixing in Jiangsu province, Guangde in Anhui province, Anji and Lishui in Zhejiang province,36 fugual strains were isolated from fruiting badies of S. bambusicola. Two molecular phylogenetic trees were generated by neighbor-joining method and maximum parsimony method with the internal transcribed spacers (ITS) of rDNA sequences of the 36 strains and 15 relevant sequences from GenBank. Phylogenetic analysis showed that 29 strains belonged to Ascomycota, 3 strains belonged to Basidiomycota, and 4 stains belonged to Deuteromycotina. In Ascomycota, the members of the Pleosporales and Apiosporaceae were dominant groups, secongdly including the Hypocreales and Sordariales.更多还原