1、Research paper 研究论文 22 January 2023,42(1):344-352 菌物学报 Mycosystema ISSN1672-6472 CN11-5180/Q Doi:10.13346/j.mycosystema.220357 资助项目:国家自然科学基金(31901696,81803393);广东省自然科学基金(2022A1515011066,2021A1515010960);广州市科技计划项目(202002030225,202102021281);广东省科技计划项目(210909154531306);广东省扬帆计划(2017YT05S115)This work wa
2、s supported by the National Natural Science Foundation of China(31901696,81803393),the Natural Science Foundation of Guangdong Province(2022A1515011066,2021A1515010960),the Science and Technology Program of Guangzhou(202002030225,202102021281),the Science and Technology Special Project of Guangdong
3、Province(210909154531306),and the Guangdong Province Sail Plan(2017YT05S115).*Corresponding authors.E-mail:;#Co-first author Received:2022-09-20;Accepted:2022-10-20 菌物学报 Copyright 2023 Institute of Microbiology,CAS.All rights reserved.| Http:/journals- Tel:+86-10-64807521 344 互补交配型配对诱导蛹虫草有性子实体形成 冯德龙
4、1,2#,胡惠萍2#,雍天乔2*,刘远超2,肖春2,黄龙花2,谢意珍2,吴清平2*1 黄山风景区管理委员会园林局,安徽 黄山 245800 2 广东省科学院微生物研究所 华南应用微生物国家重点实验室 广东省微生物安全与健康重点实验室 农业农村部农业微生物组学与精准应用重点实验室,广东 广州 510070 摘 要:前期我们获得了优良性状蛹虫草 Cordyceps militaris 野生菌株 W141436,其子实体虫草素含量高达 3.72 mg/kg 干重,子实体多糖高达 6.7 g/100 g 干重,但在大规模应用中发现它发生退化。针对蛹虫草人工栽培退化问题,我们提出蛹虫草子实体形成是有性生
5、殖过程,其两种交配型发生交配是蛹虫草子实体形成的必要条件。本文基于交配型基因分子标记研究了优良性状蛹虫草野生菌株W141436 的退化机制。具体地,采用单孢子分离的方法对蛹虫草野生分离株 W141436 的子囊孢子进行分离,得到了 72 个单孢菌株。进一步采用 PCR 方法对单孢菌株及亲本菌株进行了交配型基因类型鉴定。在72个单孢菌株中,28株为Mat1-1类型交配基因型,31株为Mat1-2类型交配基因型,13 株含有与亲本相同的交配型基因。根据鉴定结果,对 2 株 Mat1-1 型(SP28、SP33)和 2 株 Mat1-2型(SP15、SP32)菌株进行了栽培实验。结果表明,形成子实体
6、的栽培用亲本菌株同时含有 Mat1-1和 Mat1-2两种交配类型基因,即只有含不同交配型基因的菌株具有发育为子实体的能力,而含同种交配型基因的菌株则不能发育为子实体。本研究为防止蛹虫草在大规模种植中退化提供了理论依据。关键词:蛹虫草;交配型基因;分子标记;退化机制 引用本文 冯德龙,胡惠萍,雍天乔,刘远超,肖春,黄龙花,谢意珍,吴清平,2023.互补交配型配对诱导蛹虫草有性子实体形成.菌物学报,42(1):344-352 Feng DL,Hu HP,Yong TQ,Liu YC,Xiao C,Huang LH,Xie YZ,Wu QP,2023.Induction of sexual fru
7、iting-body formation by pairing the opposite mating-type isolates of Cordyceps militaris.Mycosystema,42(1):344-352 Research paper 22 January 2023,42(1):344-352 Mycosystema ISSN1672-6472 CN11-5180/Q 菌物学报 345Induction of sexual fruiting-body formation by pairing the opposite mating-type isolates of Co
8、rdyceps militaris FENG Delong1,2#,HU Huiping2#,YONG Tianqiao2*,LIU Yuanchao2,XIAO Chun2,HUANG Longhua2,XIE Yizhen2,WU Qingping2*1 Bureau of Landscape and Forestry of the Management Committee of Huangshan Mountain,Huangshan 245800,Anhui,China 2 Guangdong Provincial Key Laboratory of Microbial Safety
9、and Health,State Key Laboratory of Applied Microbiology Southern China,Laboratory of Agricultural Microbiomics and Precision Application of the Ministry of Agriculture and Rural Affairs,Institute of Microbiology,Guangdong Academy of Sciences,Guangzhou 510070,Guangdong,China Abstract:A wild strain Co
10、rdyceps militaris W141436 obtained previously with merits of high cordycepin content reaching 3.72 mg/kg(dried weight)and high polysaccharide content reaching 6.7 g/100 g(dried weight)in fruit bodies was found to be degraded in large scale cultivations.We hypothesized that mating is a process of sex
11、ual reproduction and an essential prerequisite for the fruit body development of C.militaris.This study focused on the degradation of C.militaris based on mating type gene markers.In total,72 single spore strains were isolated from C.militaris W141436 ascospores according to single spore isolating m
12、ethod.The mating-type genes of the obtained single spore strains and parent strains were identified by PCR and the results showed that 28 strains were Mat1-1(MAT-alpha)type,31 Mat1-2(MAT-HMG)and 13 parental type in the obtained 72 single spore strains.Pairing cultivation of two strains with Mat1-1 t
13、ype(SP28,SP33)and two strains with Mate1-2 type(SP15,SP32)demonstrated that cross-mating between the strains with opposite mating type genes produced well-developed fruit bodies,while cross-mating between the same mating type isolates did not produce fruit bodies.This research provided a theoretical
14、 basis for preventing C.militaris from degradation in the large scale production.Keywords:Cordyceps militaris;mating type gene;molecular markers;degradation mechanism 蛹虫草 Cordyceps militaris(L.)Link,又名北冬虫夏草、北虫草、蛹草、蛹草菌。根据系统发育分类,蛹虫草隶属于子囊菌门 Ascomycota、肉座菌目 Hypocreale、虫草菌科 Ordycipitaceae、虫草属 Cordyceps(P
15、hull et al.2022)。因蛹虫草中含有虫草素、虫草酸、腺苷及多糖等成分而具有显著的抗菌、抗肿瘤、降血糖、降尿酸和抗炎等作用(Yong et al.2016;Liu et al.2019;Rupa et al.2020;Phull et al.2022;Sun et al.2022),其化学成分和次级代谢产物与冬虫夏草相似(Nxumalo et al.2020;Liu et al.2021),其野生菌种在安徽黄山、安徽天堂寨、湖北神农架、四川海螺沟和吉林左家等地均有分布(冯德龙等 2017;郭婷等 2022),并且子实体可以大量人工栽培,因此逐渐成为了冬虫夏草的替代品,人工栽培蛹虫草,
16、具有投资少、见效快、效益高和生长周期短等优点,属种植业中的“短、平、快”项目,适宜普及推广,且鲜品需求量不断增加,备受人们关注并成为国内外研究热点(戴玉成 2022)。然而,目前在人工栽培过程中,蛹虫草菌种退化问题普遍且严重,给规模化栽培带来极大的风险,也阻碍了 冯德龙 等/互补交配型配对诱导蛹虫草有性子实体形成 研究论文 菌物学报 346 蛹虫草产业化进一步发展(怀美玉等 2022)。其中,李美娜等(2003)采用PCR-RFLP和RAPD方法对野生驯化蛹虫草及其退化菌种在基因水平进行了分析,结果表明退化菌株较野生驯化菌株在 DNA 水平上发生了频率较高的突变,这些突变可能与导致菌种退化的基因直接相关联。林清泉等(2010)研究了蛹虫草退化菌株与正常菌株在交配型、dsRNA 感染、色度和脱氢酶活性的特征差异性,表明退化菌株的脱氢酶活性和在色度培养基中的脱色能力都明显比正常菌株要弱,并建议采用脱氢酶活性和在色度培养基中的脱色能力作为鉴别退化菌株的指标。怀美玉等(2022)对蛹虫草退化研究指出显微观察菌丝是否出现粘连打结及氮蓝四唑NBT 检测菌丝活性氧含量作为蛹虫草菌株退化的检测手段。虫