利用亚硝基胍诱变和高通量测序技术分析疯草内生真菌产SW生物合成通路

doi:10.11843/j.issn.0366-6964.2019.01.020

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (1): 169-182.doi: 10.11843/j.issn.0366-6964.2019.01.020

利用亚硝基胍诱变和高通量测序技术分析疯草内生真菌产SW生物合成通路

郝宝成¹, 宋向东^1,2, 高艳^1,2, 王学红¹, 刘宇¹, 陈柯源¹, 梁妍¹, 胡毓瑶¹, 邢小勇², 温峰琴², 胡永浩^2*, 梁剑平^1*

1. 中国农业科学院兰州畜牧与兽药研究所, 农业部兽用药物创制重点实验室, 甘肃省新兽药工程重点实验室, 兰州 730050;
2. 甘肃农业大学动物医学院, 兰州 730070

收稿日期:2018-06-25 出版日期:2019-01-23 发布日期:2019-01-23
通讯作者: 胡永浩,主要从事畜禽传染病学及病原分子生物学、动物防疫与检疫研究,E-mail:yhh0817@126.com;梁剑平,主要从事兽药化学合成和中草药的提取及药理研究,E-mail:liangjp100@sina.com
作者简介:郝宝成(1983-),男,甘肃古浪人,博士,助理研究员,主要从事兽用天然药物的创制与开发研究,E-mail:haobaocheng@sina.cn;宋向东(1990-),男,甘肃天水人,硕士生,主要从事病原微生物与分子生物学研究,E-mail:18298349782@163.com
基金资助:
中国农业科学院2018年重大产出科研选题项目（CAAS-ZDXT2018008）；中国农业科学院“兽用天然药物”创新团队项目；中央级公益性科研院所基本科研业务费专项资金项目

Analysis of the Biosynthesis Pathway of Produced SW by Endophytic Fungi from Locoweed Using Nitrosoguanidine Mutation and High Throughput Sequencing Technology

HAO Baocheng¹, SONG Xiangdong^1,2, GAO Yan^1,2, WANG Xuehong¹, LIU Yu¹, CHEN Keyuan¹, LIANG Yan¹, HU Yuyao¹, XING Xiaoyong², WEN Fengqin², HU Yonghao^2*, LIANG Jianping^1*

1. Key Laboratory of New Animal Drug Project, Gansu Province, Key Laboratory of Veterinary Pharmaceutics Discovery, Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agriculture Sciences, Lanzhou 730050, China;
2. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2018-06-25 Online:2019-01-23 Published:2019-01-23

摘要/Abstract

摘要：

阐明产苦马豆素（swainsonine，SW）疯草内生真菌Alternaria Section Undifilum oxytropis中SW的生物合成通路，可以为生物发酵获得大量SW用于抗肿瘤应用和疯草的脱毒育种奠定理论基础和技术支撑。采用不同作用剂量的化学诱变剂亚硝基胍，在28℃，120 r·min^-1与Alternaria Section Undifilum oxytropis孢子悬液分别作用5、10、15、20、25、30 min，选取生长良好的菌株接种PDA培养基，连续培养发酵24 d，应用α-甘露糖苷酶活性分析法检测菌丝及发酵液中SW含量，评价其传代及产SW稳定性。联合使用玻璃珠、液氮破壁法预处理菌丝，选用真菌基因组提取试剂盒提取原始菌株和突变株D4基因组DNA，经纯化、酶切、构建菌株DNA文库，并应用荧光定量PCR法检测评价文库构建质量，利用Ion torrent PGM^TM测序平台进行突变株和原始株高通量全基因组测序。结果表明，经化学诱变成功获得1株突变株D4，利用MIRA软件对高通量测序序列拼接、组装，分别获得突变株D4和原始株相关基因组片段19、21条，经BLAST、GO分析，注释到Alternaria Section Undifilum oxytropis生物合成SW的关键基因SwnK、SwnH2、SwnH1、SwnR和关键酶putative amino acid transporter、pyrroline-5-carboxylate reductase、formate/glycerate dehydrogenase catalytic、saccharopine reductase-like protein，并结合KEGG数据库，预测了Alternaria Section Undifilum oxytropis产SW可能的生物合成通路。本研究为揭示Alternaria Section Undifilum oxytropis生物合成SW机制作出了新的探索尝试。

Abstract:

The elucidation of the producing swainsonine biosynthesis pathway of Alternaria Section Undifilum oxytropis, an endophytic fungi isolated from locoweed, which can provide theoretical basis and technical support for the biological fermentation to obtain a large number of SW for anti-tumor applications and detoxification breeding of locoweed. Used different doses of nitrosoguanidine, reacted with spore suspension of Alternaria Section Undifilum oxytropis at 120 r·min^-1 for 5, 10, 15, 20, 25, and 30 min at 28℃, respectively, and selected good growth strains were inoculated with PDA medium. The culture was continued for 24 days, and the α-mannosidase inhibition method was used to detect SW content in the mycelia and the fermentation broth to evaluate the genetic stability of growth. The mycelium was pretreated with glass beads and liquid nitrogen disruption method. The genomic DNA extraction kit was used to extract the genomic DNA of the original strain and the mutant strain D4. The purified and endonuclease-strained DNA library was constructed and detected by fluorescent quantitative PCR. Evaluate library construction quality and use the Ion torrent PGM^TM sequencing platform to complete high-throughput whole-genome sequencing of mutant and original strains. The results showed that a mutant D4 was successfully obtained by chemical mutagenesis. The high-throughput sequencing technology was spliced and assembled using MIRA software, and 19 and 21 related genomic fragments of mutant D4 and original strain were obtained respectively. The experimental results were analyzed by BLAST and GO software, and annotation to the key synthesis genes of SW in Alternaria Section Undifilum oxytropis, such as SwnK, SwnH2, SwnH1, SwnR putative amino acid transporter, etc., and the key synthesis enzymes, such as pyrroline-5-carboxylate reductase, formate/glycerate dehydrogenase catalytic, saccharopine reductase-like protein, combined with KEGG database, the possible biosynthetic pathway of SW is predicted in Alternaria Section Undifilum oxytropis. The above results will contribute to clarify the SW biosynthesis mechanism of Alternaria Section Undifilum oxytropis.

中图分类号:

S852.66

郝宝成, 宋向东, 高艳, 王学红, 刘宇, 陈柯源, 梁妍, 胡毓瑶, 邢小勇, 温峰琴, 胡永浩, 梁剑平. 利用亚硝基胍诱变和高通量测序技术分析疯草内生真菌产SW生物合成通路[J]. 畜牧兽医学报, 2019, 50(1): 169-182.

HAO Baocheng, SONG Xiangdong, GAO Yan, WANG Xuehong, LIU Yu, CHEN Keyuan, LIANG Yan, HU Yuyao, XING Xiaoyong, WEN Fengqin, HU Yonghao, LIANG Jianping. Analysis of the Biosynthesis Pathway of Produced SW by Endophytic Fungi from Locoweed Using Nitrosoguanidine Mutation and High Throughput Sequencing Technology[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 169-182.

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利用亚硝基胍诱变和高通量测序技术分析疯草内生真菌产SW生物合成通路

Analysis of the Biosynthesis Pathway of Produced SW by Endophytic Fungi from Locoweed Using Nitrosoguanidine Mutation and High Throughput Sequencing Technology

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