一株酵母菌株的鉴定(论文15000字)
摘要
酵母是一种在工业生产中广泛应用的菌株，对酵母的鉴定和分类可以很好的指导研究者开展各项相关的基础研究以及应用研究。传统酵母菌分类以形态学为主，但精确性较差。本论文拟通过基于生理生化实验的Biolog 微生物自动鉴定系统和基于18S rRNA基因和26S rRNA基因序列的分子生物学方法对实验室分离保藏的一株酵母菌株NJ730进行系统鉴定。结果显示，Biolog微生物自动分析系统实验中NJ730可利用碳源麦芽糖，麦芽三糖，松三糖，异麦芽酮糖，蔗糖，海藻糖，松二糖，葡萄糖，半乳糖等，系统数据分析显示本次鉴定的SIM值 = 0. 967 > 0.75；DIS值 = 0.471 <5. 0；PROB为0.999，并将NJ730鉴定为一株酿酒酵母。通过PCR方法将酵母菌株NJ730的26 S rRNA基因片段和18S rRNA基因片段扩增出并送测序，将测序所得的基因序列与NCBI数据库中的同源性序列进行比对，并构建系统进化树。由系统进化树可知，可将菌株NJ730鉴定为一株酿酒酵母（Saccharomyces cerevisiae），与上述几种鉴定方法结果一致。综上所述，通 过几种不同方法，将菌株NJ730确定鉴定为一株酿酒酵母（Saccharomyces cerevisiae）。
关键词：酵母 分类鉴定 Biolog 18S rRNA基因 26S rRNA基因
Identification of a yeast strain
Abstract
Yeast is a kind of widely used strains in mordern industry. Identification and classification of yeast can help researchers with the related basic research and application research. Traditional classification of yeast is based on the morphology, but the accuracy is poor. In this study, Biolog microbial identification system based on physiological and biochemical tests and molecular biology methods based on the 18S rRNA gene and 26S rRNA gene sequence was employed to identify a yeast strain NJ730 isolated by our laboratory. The results of Biolog system showed that NJ730 can use maltose, maltotriose, melezitose, isomaltulose, sucrose, trehalose, turanose, glucose, galactose etc. as the sole carbon source. In addition, the system data analysis showed that the data of SIM = 0.967 > 0.75; DIS = 0.471 <5. 0; PROB 0.999 in this test, which was in good accordance with Saccharomyces cerevisiae. Next, the 26S rRNA and 18S rRNA gene fragment of was was amplified and sequenced, which were aligned with the homology gene sequences in NCBI database. Finally, the phylogenetic trees based on the alignments were built, which showed that the strain NJ730 shared 100 % sequence identity of its 26S rRNA and 18S rRNA gene sequences with Saccharomyces cerevisiae. In conclusion, strain NJ730 was identified as Saccharomyces cerevisiae through morphological features, physiological and biochemical characteristics, as well as phylogenetic analysis of 26S rRNA and 18S rRNA gene sequences.
Key Words: yeast; identification and classification; Biolog; 18S rRNA gene; 26S rRNA gene
目 录
摘要 I
Abstract II
第一章 文献综述 1
1.1引言 1
1.2酵母菌的鉴定方法 1
1.2.1常规分类学方法 1
1.2.2化学分类学方法 2
1.2.3分子分类学方法 3
1.2.4其他分子生物学鉴定方法 6
1.3 Biolog 微生物自动鉴定系统 6
1.4本课题研究的目的和意义 7
第二章 材料与方法 8
2.1 实验材料 8
2.1.1 菌株和质粒 8
2.1.2 实验试剂及耗材 8
2.1.3 实验仪器 9
2.1.4 实验所用培养基和溶液 10
2.2 实验方法 10
2.2.1 酵母镜检实验 10
2.2.2酵母菌的Biolog鉴定 10
2.2.3酵母菌的分子生物学鉴定 11
第三章 结果与分析 16
3.1 酵母菌株NJ730的显微镜观察 16
3.2 酵母菌株NJ730的Biolog鉴定 17
3.3 酵母菌株NJ730的分子生物学鉴定 20
3.3.1酵母菌株NJ730基因组DNA提取 20
3.3.2 酵母菌株NJ730的26S rRNA基因的PCR扩增 21
3.3.3 酵母菌株NJ730的18S rRNA基因的PCR扩增 22
3.3.4 26S rRNA和18S rRNA基因的PCR扩增产物切胶回收纯化 23
3.3.5 切胶回收产物的酶连、转化 24
3.3.6 酵母菌株NJ730的26S rRNA基因序列 24
3.3.7 酵母菌株NJ730的18S rRNA基因序列 25
3.3.8 酵母菌株NJ730的26S rRNA基因序列比对分析 26
3.3.9 酵母菌株NJ730的18S rRNA基因序列比对分析 27
3.3.10 酵母菌株NJ730的基于26S rRNA基因序列的系统进化树 28
3.3.11 酵母菌株NJ730的基于18S rRNA基因序列的系统进化树 29
第四章 结论与展望 30
4.1 结论 30
4.2 展望 31
参考文献 32
致谢 34
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