摘 要：本实验以桦褐孔菌菌核为实验材料，从中分离纯化出8种菌核内微生物。将分离出的8种菌株放置恒温培养箱中培养，数天后对菌株的菌落形态进行观察，在显微镜下观察菌株的产孢结构并拍照记录。再将这8种菌株用植物基因组DNA提取试剂盒提取DNA,对其ITS序列进行PCR扩增，所获取的基因序列与( http://blast。ncbi。nlm。nih。gov/Blast。cgi)数据库中调出的相似性较高的相关菌株的基因序列进行比对分析，最终确定其归属。结果表明8种内生菌生长均比较缓慢，其中菌A生长最迅速，菌C, F, J, L较为缓慢，菌O和Q几乎未生长。另外，参照ITS测序结果，菌株A和Dendrothyrium longisporum( GENEbank登录号：JX496097)相似度达99 %，初步确定菌株A为Dendrothyrium longisporum;菌株C和 Pyrenochaeta sp。( GENEbank登录号：KF660551。1）刺壳孢菌的比对结果小于95 %；菌株F和Coniothyrium carteri(GENEbank登录号：KF251209)比对结果为99 %，初步确定菌株F为Coniothyrium carteri；菌株J和Phialophora dancoi(GENEbank登录号：AB190869)瓶霉属的比对结果为99 %,初步确定菌株J为Phialophora dancoi;菌株M和Cyphellophora pauciseptata( GENEbank登录号：NR_132868)无柄孢菌的比对小于90 %；菌株L和菌株O比对结果和菌株C相同；菌株Q和Cryptosporiopsis persispora(GENEbank登录号：JF714251)的比对结果小于95 %。本研究旨在对桦褐孔菌菌核内微生物进行分离和形态观察，然后对分离的微生物进行DNA提取、ITS序列测序和比对，并构建系统树。从而初步鉴定桦褐孔菌菌核内微生物的种属，为桦褐孔菌和微生物的研究做准备,并为大型真菌中存在内生微生物提供实际证据，为寻找和开发野生菌种资源提供新的途径。76466

毕业论文关键词：桦褐孔菌，分离纯化，提取DNA, PCR扩增，ITS序列测序，初步鉴定。

Isolation and identification of microorganism in Sclerotia of inonotus obliquus

Abstract: This experiment chose sclerotia of Inonotus obliquus as material and abstracted 8 sclerotia within organisms。 Placed the 8 strains into the constant temperature incubator, observed the characteristics of colony morphology several days later, then observed the sporing structure under a microscope and took pictures to record them。 Used the plant genomic DNA kit to extract DNA from the 8 strains。 PCR amplification of there ITS sequence。 Compared and analysed between sequence of the strains with highly related from NCBI database( http://blast。ncbi。nlm。nih。gov/Blast。cgi) and sequence obtained。 Finally determine the attribution of the 8 strains。 Results show that all of the 8 strains grow slowly, in which strain A grew most rapidly, strain C, F, J, L relatively slow, but O and Q almost not grew。 In addition, referred to the results of ITS sequencing, strain A and Dendrothyrium longisporum(GENEbank, Locus No。 JX496097) were similar to 99%, initially identified strain A is Dendrothyrium longisporum; strain C and Pyrenochaeta sp。 ( GENEbank, Locus No。 KF660551。1) were less than 95%; strain F and Coniothyrium carteri( GENEbank, Locus No。 KF251209) were similar to 99%, initially identified strain F is Coniothyrium carteri; strain J and Phialophora dancoi( GENEbank, Locus No。 AB190869) were similar to 99%, initially identified strain F is Coniothyrium carteri; strain M and C yphellophora pauciseptata( GENEbank, Locus No。 NR_132868) were less than 90%;strain L and O were same to strain C; strain Q and Cryptosporiopsis persispora( GENEbank, Locus No。 JF714251) were less than 95%。 This research aimed at separating microorganism in sclerotia of Inonotus obliquus and observing their morphology, then extracted DNA from the microorganism, sequenced the ITS sequence and compared。 Constructing the phylogenetic trees to preliminary identified the spicies of these microorganisms。 Then we preliminary identified species of microorganisms in sclerotia of Inonotus bliquus, prepared for the study of Inonotus obliquus and microorganisms。 Besides, provided evidence that there did have endophytic microbes in macrofungus。 Finally, provided new ways to search for and exploit wild strain resource。