宁夏苦豆子中产苦参碱内生真菌的分离与鉴定  

Isolation and Identification of Matrine-Producing Fungal Endophytes from Sophora alopecuroides in Ningxia

作　　者：余永涛[1] 何生虎[1] 赵清梅[2] 

机构地区：[1]宁夏大学农学院,银川750021 [2]北方民族大学生物科学与工程学院,银川750021

出　　处：《中国农业科学》2013年第13期2643-2654,共12页Scientia Agricultura Sinica

基　　金：宁夏高等学校科学研究项目(2011-4110288);国家自然科学基金项目(31201962);宁夏自然科学基金项目(NZ1118);宁夏大学自然科学基金项目(ndzr09-12)

摘　　要：【目的】从宁夏野生苦豆子中分离内生真菌,筛选能够产生苦参碱、氧化苦参碱、氧化槐果碱或槐定碱4种喹诺里西啶类生物碱的内生真菌。【方法】应用光学显微镜和扫描电子显微镜对分离菌株显微形态进行观察,根据形态对各菌株进行初步分类;应用薄层色谱法、气相色谱-质谱联用法和酸性染料比色法检测各分离菌株的生物碱成分。对能够产生生物碱的真菌ITS和SSU rDNA序列进行扩增和测序,根据序列信息进行同源性比较和系统发育树构建;结合形态特征,对其进行种属分类。【结果】从苦豆子中共分离到27株真菌,其中23株分属于青霉属、曲霉属、链格孢属、镰刀菌属、Simplicillium属真菌,4株为不产孢菌;筛选出E1、E3和E5这3株能够产生苦参碱的内生真菌,均属于Simplicillium属真菌,其菌丝中苦参碱的含量分别为20.4、17.4和37.6μg.g-1。【结论】根据真菌形态特征、同源性比较和遗传进化分析结果,确定该3株产苦参碱内生真菌均为Simplicillium lanosoniveum。[ Objective ] An experiment was carried out to isolate and confirm whether the presence of fungal endophyte that could produce quinolizidine alkaloids including matrine, oxymatrine, oxysophocarpine or sophoridine from wild Sophora alopecuroides L in Ningxia. [Method] The isolates were observed under the light microscope and scanning electron microscopy, respectively. The classification of each isolate was performed preliminarily according to the morphological characteristics. The chemical compositions of isolated fungi were detected by thin-layer chromatography, gas chromatograph-mass spectrometer and acid dye colorimetry to screen the quinolizidine alkaloids-producing fungal endophytes. The ITS and SSU rDNA sequences of the fungi were amplified and sequenced. Homology comparison was conducted and phylogenetic tree was established based on the sequences of the fungi. The alkaloids-producing fungal endophytes were classified according to the fungal morphology and phylogenetic analysis. [Result] Twenty-seven fungi were isolated from S. alopecuroides. Twenty-three of the 27 isolates were classified as Penicillium, Aspergillus, Alternaria and Fusarium based on morphology of conidia. The other four isolates could not produce conidia and were classified as mycelia sterile. However, only three fungal isolates, El, E3 and E5 that belonged to Simplicillium, were conformed contain substantial amounts of matrine which is a kind of quinolizidine alkaloids. The contents of matrine were 20.4 p.g.g-l, 17.4 p.g.g-1 and 37.6 p.g.g-1, respectively, in the mycelia of El, E3 and E5. [ Conclusion]Homology and phylogenetic analysis results showed that the endophytes are more closely related to Simplicillium lanosoniveum. The morphologies of conidiophores and conidia in the three fungal endophytes were also highly similar to S. lanosoniveum. The results indicated that El, E3 and E5 were S. lanosoniveum.

关 键 词：苦豆子 苦参碱 内生真菌 Simplicillium lanosoniveum 

分 类 号：S567.3[农业科学—中草药栽培]