Xu Li-Li, Ling Xiao-Feng, Zhao Shu-Juan, Wang Ru-Feng, Wang Zheng-Tao
Institute of Science, Technology and Humanities, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
MOE Key Laboratory for Standardization of Chinese Medicines, SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Chin Herb Med. 2020 Jun 26;12(3):297-302. doi: 10.1016/j.chmed.2020.03.007. eCollection 2020 Jul.
In the present study, was screened for fungi communities to clarify their diversity and community assemblage in hosts. Meanwhile, the identification and activity assays of the strains were also conducted.
By culture-dependent (endophytic fungi isolations from plant sections) and culture-independent (metagenomic library and cloning from plant sections) techniques, fungi communities were studied. The metagenomic library was generated using direct DNA isolation of whole plants, plant radixes, plant stems, plant leaves, plant flowers and soils around the plant. Meanwhile, endophytes were isolated from all parts of plants. After fermentation of the fungi isolations, all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines (HCT116, BEL7404, A549, MDA-MB-231).
Eventually, 200 strains were isolated and 103 strains were further identified through the internal transcribed spacer (ITS, ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4. A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units (OTU) were identified by 454 pyrosequencing. Through 454 pyrosequencing, the main fungal genera were , , , and and the major fungal genera which were directly isolated were and . Activity assays showed strains 5-26 ( sp.) and 6-2 () had the outstanding cytotoxicity to all the tested cell lines with IC values <5 μg/mL.
This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in by high-throughput 454 sequencing technology and fungi isolation methods. Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.
在本研究中，对[具体研究对象]进行真菌群落筛选，以阐明其在宿主中的多样性和群落组成。同时，还对菌株进行了鉴定和活性测定。
通过依赖培养（从植物组织中分离内生真菌）和不依赖培养（宏基因组文库构建及从植物组织中克隆）技术研究真菌群落。宏基因组文库利用全株植物、植物根系、植物茎、植物叶、植物花及植物周围土壤的直接DNA提取构建。同时，从[具体植物]的各个部位分离内生菌。真菌分离物发酵后，评估所有分离物对四种人类癌细胞系（HCT116、BEL7404、A549、MDA-MB-231）的细胞毒性。
最终分离出200株菌株，通过使用通用引物ITS5和ITS4对内部转录间隔区（ITS，ITS1和ITS2区域）序列进行进一步鉴定，确定了103株。通过454焦磷酸测序鉴定出总共59,106条真菌序列，对应374个假定的操作分类单元（OTU）。通过454焦磷酸测序，主要真菌属为[具体属名1]、[具体属名2]、[具体属名3]、[具体属名4]和[具体属名5]，直接分离得到的主要真菌属为[具体属名6]和[具体属名7]。活性测定表明，菌株5 - 26（[具体种名1]）和6 - 2（[具体种名2]）对所有测试细胞系具有显著的细胞毒性，IC值<5μg/mL。
本研究通过高通量454测序技术和真菌分离方法揭示了[具体研究对象]中丰富的内生真菌资源和多种遗传信息。活性测定表明内生菌是潜在抗癌药物的有前景的天然来源。
