花脸香蘑JZ01菌株分子生物学鉴定及其发酵液抗菌活性研究

发布时间：2018-06-26 01:42

本文选题：花脸香蘑 + RAPD　；参考：《吉林大学》2017年硕士论文

【摘要】：花脸香蘑是一种名贵的食用药用菌,具有较高的开发价值,野生菌数量少、产量低,导致其价格昂贵。花脸香蘑JZ01子实体采集于吉林大学珠海学院实验楼草坪,由化学与药学系王兰英老师鉴定并命名为花脸香蘑JZ01。本文通过RAPD(随机扩增多态性DNA标记,Random Amplified Polymorphic DNA)、ITS(核糖rDNA内部转录间隔区,Internal Transcribed Spacer)序列分析对花脸香蘑JZ01菌株进行基因鉴定,并对其进行发酵液抑菌活性研究。本文采用CTAB手提法(十六烷基三甲基溴化铵法,cetyltrimethylammonium Ammonium Bromide)提取5种不同产地花脸香蘑菌丝体及子实体的基因组DNA(脱氧核糖核苷酸,deoxyribonucleic acid)并进行RAPD扩增,采用上海生工生物工程公司的12个引物对5个样品进行PCR(聚合酶链式反应,Polymerase Chain Reaction)扩增,并从12个引物的检测图中选出8个条带清晰的引物进行数据分析,8个引物在5个样品中获得46条扩增片段。用NTSYS软件计算样品间的遗传相似系数(GS值),得到供试材料遗传相似矩阵。分析得出5个样品的GS值范围为:0.2826-0.9130,变幅为0.6304。根据遗传相似系数越大,亲缘关系越近,遗传相似系数越小,表明亲缘关系越远的规律。得出结论:4号样品和5号样品之间的遗传相似系数最大为0.9130,表明这两个样品之间的亲缘关系较近,遗传差异性小。3号样品(JZ01)和4、5号之间的遗传相似系数最小,分别为0.2826,表明花脸香蘑JZ01同4、5号样品之间的亲缘关系较远,遗传差异性较大,存在较高的遗传多样性[1]。ITS片段的序列分析结果表明:5个样品间的遗传距离在0.00460-0.06752之间,变幅为0.06292。其中4号样品和5号样品之间的遗传距离最小为0.00460,表明这两个样品之间的亲缘关系较近,遗传差异性小。1号样品和5号之间的遗传距离最大,表明1号同5号样品之间的亲缘关系较远,遗传差异性较大,存在较高的遗传多样性。通过MEGA软件聚类分析发现5种样品的亲缘关系与RAPD结果基本相吻合,说明用这两种方法鉴定花脸香蘑的亲缘关系都是可行的。在花脸香蘑发酵液的抗菌活性研究中,用不同的有机溶剂对花脸香蘑JZ01的发酵液进行萃取,实验结果表明:提取的花脸香蘑胞外小分子乙酸乙酯相对所选菌种的生长均有较好的抑制作用,尤其是对粘质沙雷氏菌的抑制性最好。而花脸香蘑胞外小分子正丁醇相的抗菌效果不好,不宜采用。花脸香蘑JZ01胞外小分子乙酸乙酯相对革兰氏阳性菌(G+)、革兰氏阳性菌(G-)均有一定的抑制作用,说明花脸香蘑JZ01具有广泛的抗菌活性,可作为天然抑菌剂加以开发应用。
[Abstract]:Tricholoma comatus is a rare edible medicinal fungus with high development value, low quantity of wild fungus and low yield, which leads to its high price. JZ01 fruiting entity was collected from the lawn of the experimental building of Zhuhai College of Jilin University. It was identified and named JZ01by Mr. Wang Lanying, Department of Chemistry and Pharmacy. The strain JZ01 was identified by random Amplified polymorphic DNA (random Amplified polymorphic DNA) its (ribosomal rDNA internal transcriptional spacer) sequence analysis and its antibacterial activity was studied. In this paper, cetyltrimethylammonium Ammonium bromide (CTAB) method was used to extract genomic acid (deoxyribonucleic acid) from mycelia and fruiting bodies of Tricholoma comatus from five different habitats. Five samples were amplified by PCR (Polymerase chain reaction) using 12 primers of Shanghai Bioengineering Company. Eight primers with clear bands were selected from 12 primer detection maps for data analysis, and 46 amplified fragments were obtained from 5 samples by 8 primers. The genetic similarity coefficient (GS value) between samples was calculated by NTSYS software, and the genetic similarity matrix of tested materials was obtained. The results show that the GS values of the 5 samples are in the range of: 0.2826-0.9130, with a range of 0.6304. The larger the genetic similarity coefficient is, the closer the genetic relationship is and the smaller the genetic similarity coefficient is, which indicates that the farther the genetic similarity coefficient is, the farther the genetic similarity coefficient is. Conclusion: the maximum genetic similarity coefficient between sample 4 and sample 5 is 0.9130, which indicates that the genetic relationship between the two samples is close and the genetic difference is small. The genetic similarity coefficient between sample 3 (JZ01) and sample 45, 5 is the smallest. It was 0.2826, respectively, which indicated that the genetic relationship between JZ01 and samples No. 4 and No. 5 was far, the genetic diversity was higher, and the sequence analysis of its fragment showed that the genetic distance of the five samples ranged from 0.00460-0.06752, and the variation was 0.06292.The results showed that the genetic distance of the five samples was between 0.00460-0.06752 and 0.06292. The genetic distance between sample No. 4 and sample No. 5 was 0.00460, which indicated that the genetic relationship between the two samples was close and the genetic difference was small. The genetic distance between sample No. 1 and sample No. 5 was the largest. The results showed that the genetic relationship between 1 and 5 was far, genetic diversity was higher and genetic diversity was higher. Cluster analysis of Mega software showed that the genetic relationships of five samples were basically consistent with RAPD results, which indicated that it was feasible to use these two methods to identify the genetic relationship of Tricholoma comatus. In the study of antimicrobial activity of fermentation broth of Tricholoma comatus, different organic solvents were used to extract the fermentation broth of JZ01. The experimental results showed that the extracellular small molecule ethyl acetate of the extracted Tricholoma comatus had a good inhibitory effect on the growth of the selected strains, especially on the mucinous Shareh's bacteria. The antimicrobial effect of butanol phase is not good and should not be used. Compared with Gram-positive bacteria (G) and Gram-positive bacteria (G-), the extracellular ethyl acetate of JZ01 has certain inhibitory effect, which indicates that JZ01 has extensive antibacterial activity and can be used as a natural bacteriostatic agent.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S646

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