本文選題：花臉香蘑 + RAPD�。� 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：花臉香蘑是一種名貴的食用藥用菌,具有較高的開發(fā)價值,野生菌數(shù)量少、產(chǎn)量低,導(dǎo)致其價格昂貴。花臉香蘑JZ01子實體采集于吉林大學(xué)珠海學(xué)院實驗樓草坪,由化學(xué)與藥學(xué)系王蘭英老師鑒定并命名為花臉香蘑JZ01。本文通過RAPD(隨機擴增多態(tài)性DNA標(biāo)記,Random Amplified Polymorphic DNA)、ITS(核糖rDNA內(nèi)部轉(zhuǎn)錄間隔區(qū),Internal Transcribed Spacer)序列分析對花臉香蘑JZ01菌株進行基因鑒定,并對其進行發(fā)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銨法,cetyltrimethylammonium Ammonium Bromide)提取5種不同產(chǎn)地花臉香蘑菌絲體及子實體的基因組DNA(脫氧核糖核苷酸,deoxyribonucleic acid)并進行RAPD擴增,采用上海生工生物工程公司的12個引物對5個樣品進行PCR(聚合酶鏈?zhǔn)椒磻?yīng),Polymerase Chain Reaction)擴增,并從12個引物的檢測圖中選出8個條帶清晰的引物進行數(shù)據(jù)分析,8個引物在5個樣品中獲得46條擴增片段。用NTSYS軟件計算樣品間的遺傳相似系數(shù)(GS值),得到供試材料遺傳相似矩陣。分析得出5個樣品的GS值范圍為:0.2826-0.9130,變幅為0.6304。根據(jù)遺傳相似系數(shù)越大,親緣關(guān)系越近,遺傳相似系數(shù)越小,表明親緣關(guān)系越遠的規(guī)律。得出結(jié)論:4號樣品和5號樣品之間的遺傳相似系數(shù)最大為0.9130,表明這兩個樣品之間的親緣關(guān)系較近,遺傳差異性小。3號樣品(JZ01)和4、5號之間的遺傳相似系數(shù)最小,分別為0.2826,表明花臉香蘑JZ01同4、5號樣品之間的親緣關(guān)系較遠,遺傳差異性較大,存在較高的遺傳多樣性[1]。ITS片段的序列分析結(jié)果表明:5個樣品間的遺傳距離在0.00460-0.06752之間,變幅為0.06292。其中4號樣品和5號樣品之間的遺傳距離最小為0.00460,表明這兩個樣品之間的親緣關(guān)系較近,遺傳差異性小。1號樣品和5號之間的遺傳距離最大,表明1號同5號樣品之間的親緣關(guān)系較遠,遺傳差異性較大,存在較高的遺傳多樣性。通過MEGA軟件聚類分析發(fā)現(xiàn)5種樣品的親緣關(guān)系與RAPD結(jié)果基本相吻合,說明用這兩種方法鑒定花臉香蘑的親緣關(guān)系都是可行的。在花臉香蘑發(fā)酵液的抗菌活性研究中,用不同的有機溶劑對花臉香蘑JZ01的發(fā)酵液進行萃取,實驗結(jié)果表明:提取的花臉香蘑胞外小分子乙酸乙酯相對所選菌種的生長均有較好的抑制作用,尤其是對粘質(zhì)沙雷氏菌的抑制性最好。而花臉香蘑胞外小分子正丁醇相的抗菌效果不好,不宜采用。花臉香蘑JZ01胞外小分子乙酸乙酯相對革蘭氏陽性菌(G+)、革蘭氏陽性菌(G-)均有一定的抑制作用,說明花臉香蘑JZ01具有廣泛的抗菌活性,可作為天然抑菌劑加以開發(fā)應(yīng)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S646

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1 張麗麗;花臉香蘑JZ01菌株分子生物學(xué)鑒定及其發(fā)酵液抗菌活性研究[D];吉林大學(xué);2017年

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