植物内生真菌的分离纯化及其代谢产物的抗肿瘤活性检测
毕业论文关键字：植物内生真菌；抗肿瘤；菌种鉴定；
The separation and purification of endophytic fungi and the antitumor activity screening of their metabolic products
Abstract: Endophytic fungi are widely distributed in nature in almost all plants as a special category of Fungi. They can produce abundant metabolites including insecticidal, antimicrobial and antitumor active substances. Part has been applied in clinical for its significant effect . 320 species of plant endophytic fungi were isolated from 32 kinds of plant specimens picking from Nantong and Anhui . The Antitumor activity of their metabolic products to A549 tumor cells in vitro were screened using MTT method . The results showed that SK11359043 has the best significant inhibitory activities to A549 cells. DNA was extracted from strain SK11359043. Its ITS was amplified and sequenced in Shanghai Sangon Biotech (Shanghai).The sequence was blasted in the NCBI database. The classification was identificated combining with the morphology of hyphae and spores of endophytic fungi. The results showed that strain SK11359043 belong to Phoma sp.
Key words: Endophytic fungi; Antitumor activity ;Species identification.
目录
引言 1
1材料与方法 2
1.1植物样品的采集 2
1.2培养基 2
1.3实验仪器 2
1.4植物内生真菌的分离和纯化 2
1.4.1 试剂 2
1.4.2取样 2
1.4.3表面灭菌 2
1.4.4平板接种 3
1.4.5室温培养 3
1.4.6纯化 3
1.5菌种保藏和发酵培养 3
1.6 MTT法检测内生真菌代谢产物的抗肿瘤活性 3
1.6.1试剂 3
1.6.2细胞培养 3
1.6.3细胞复苏 3
1.6.4细胞传代 4
1.6.5细胞冻存 4
1.6.6 MTT实验 4
1.7提取目标内生真菌的DNA 4
1.8 PCR扩增 5
1.9电泳 5
1.10菌种鉴定 6
2 结果与分析 6
2.1内生真菌分离结果 6
2.2 内生真菌代谢产物抗肿瘤活性的检测 7
2.3目标内生真菌的菌种鉴定 9
3 讨论 10
3.1 内生真菌的分离存在的问题 10
3.2 内生真菌代谢产物抗肿瘤活性的研究 12
致 谢 12
参考文献 12
引言
在自然界里，微生物和植物是息息相关、不可分离的。体现这一点的典型例子有很多，如存在豆科植物中的根瘤菌，木本植物中的菌根真菌等，它们与宿主的关系非常密切。内生真菌和植物之间相互共生，这一点从上世纪80年代被发现，主要可归纳为：（1）植物向内生真菌供给光合产物和矿物质；（2）植物的生长也离不开内生真菌的促进作用，也能降低食草动物、害虫或其他情况来威胁植物生长的风险 [1]。
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