砷氧化细菌和砷代谢基因导致冬虫夏草砷超富集机制研究

Ophiocordyceps sinensis has been widely used as tonic in Asian medicine. Unfortunately, arsenic accumulated in wild O. sinensis. Arsenic was listed in WHO 10 chemicals of major public health concern, and was confirmed as carcinogens by International Agency for Research on Cancer. According to transcriptome and genome-wide data analysis, we assumed that Herminiimonas arsenicoxydans and Arsenic metabolism related genes cause As-accumulation in O. sinensis. Two aspects following will be studied in this project: 1) H. arsenicoxydans will be purified from soil and the distribution will be confirmed. After adding As resource and H. arsenicoxydans, speciation and content of arsenic in O. sinensis will be detected by HPLC-AFS to confirm the relationships between H. arsenicoxydans and As-accumulation in O. sinensis. 2) Full-length cDNA of six key enzymes will be cloned using RACE technologies. E. coli DH5 ɑ and arsenate-sensitive yeast strain RM1 were used for expression and functional verification. Depending on the study above, we can conclude that H. arsenicoxydans and Arsenic metabolism related genes cause As-accumulation in O. sinensis.

冬虫夏草价格昂贵，但野生冬虫夏草砷含量普遍超标3-6倍，砷为致癌物，是WHO列出的引起重大公共卫生关注的10种化学品之一。本项目通过转录组和全基因组数据分析，结合申请者研究工作基础，提出砷氧化细菌和冬虫夏草砷代谢基因是砷超富集的主要原因。将重点研究：1）通过DNA分子鉴定确定砷氧化细菌在冬虫夏草及土壤中的分布；分离、纯化得到砷氧化细菌菌株；添加外源砷及砷氧化细菌，采用HPLC-AFS联用技术检测冬虫夏草菌中砷的价态和含量，确定砷氧化细菌与冬虫夏草砷超富集的关系；2）对冬虫夏草菌中与砷代谢相关的6个关键酶基因采用RACE技术进行全长克隆；构建真核表达载体后，采用砷敏感型酵母菌株RM1表达蛋白，质谱验证；通过添加外源砷，从体内及体外验证蛋白的生化特征和催化功能。揭示砷氧化细菌和砷代谢基因导致冬虫夏草砷超富集的作用机制，为冬虫夏草安全使用提供基础。

冬虫夏草是一种名贵中药材，但药材中砷含量普遍超标。从四川、西藏5个冬虫夏草菌产地土壤中分离鉴定出2个抗砷细菌XB和XNQ，XB和XNQ分别为不动杆菌属的Acinetobact guillouiae和Acinetobacter nosocomialis，均为革兰氏阴性菌。通过抗砷实验发现两种细菌分别冬虫夏草菌对As(Ⅲ)与As(Ⅴ)的吸收均有促进作用。土壤高浓度砷会促进冬虫夏草总砷的提高。通过三代转录组重测序发现，通过冬虫夏草菌对抗砷细菌与As(Ⅲ)与As(Ⅴ)处理共21个处理中，冬虫夏草菌对不同处理在基因响应层面具有显著的差异。并对其中1个关键酶基因进行了全长克隆及功能验证。因此冬虫夏草菌对As(Ⅲ)与As(Ⅴ)都有较强的吸附作用，两个抗砷细菌在高浓度砷存在的情况下会促进砷的富集。因此生产过程中应通过使用砷含量地的土壤或饲养介质以及无菌洁净环境来控制冬虫夏草中总砷的含量，实现冬虫夏草无公害生产。项目相关成果发表SCI论文1篇，国内核心期刊3篇，申请专利1项，获得广东科学技术奖一等奖1项。