沈彦辉　陈宏坤　高璐阳　徐广飞　付强强

摘要：本研究选取灰葡萄孢菌株BC 05.10为试材，克隆Bctre1基因的核苷酸序列并对其进行生物信息学分析，同时采用荧光定量PCR法研究该基因在不同侵染时期、分生孢子萌发过程中以及不同碳源、海藻糖诱导下的表达情况。结果表明：该基因ID号为5428296，全长3 341 bp，位于scaffold_17负链的495 880～499 211位置，与核盘菌Sstre1的同源关系较近; BcTRE1蛋白由1 033个氨基酸残基编码组成，该蛋白N端具有2个6发卡结构的超糖苷酶家族和1个C端糖基水解酶家族63。荧光定量PCR结果显示，随着侵染时间的延长Bctre1表达量显著增加（P<0.05），48 h后表达量增加不显著;Bctre1在分生孢子萌发24 h时表达量最高;Bctre1在海藻糖为单一碳源的培养基中表达量最高，在海藻糖诱导下12 h 后Bctre1表达量显著增加（P<0.05）。综上，Bctre1与侵染寄主过程中分生孢子萌发及不同碳源利用密切相关，在侵染后期对促进菌丝生长发挥重要作用。

关键词：灰葡萄孢;Bctre1;基因组定位;蛋白结构;表达分析

中图分类号：S432.4+4：Q78文献标识号：A文章编号：1001-4942（2020）03-0013-06

Abstract In this study， the Botrytis cinerea  strain BC 05.10 was selected as test material， and the nucleotide sequence of Bctre1 gene were cloned and analyzed by bioinformatics method. The expression of Bctre1 gene at different infection stages， in the process of conidia germination， and under different carbon sources and trehalose induction were studied by fluorescence quantitative PCR method. The results showed that the gene ID number was 5428296， and its total length was 3 341 bp， which was located at 495 880～499 211 sites of the negative chain of scaffold_17. The homologous relationship between Bctre1 and Sstre1 was close. The protein encoded by Bctre1 gene contained 1 033 amino acid residues and had two 6-hairpin structure super glucosidase family and one C-terminal glycosyl hydrolase family 63. The results of fluorescence quantitative PCR showed that the expression of Bctre1 increased significantly with the increase of infection time （P<0.05）， while the increase of Bctre1 expression after 48 h was not significant. The expression of Bctre1 was the highest when conidia germinated for 24 h and the trehalose was used as the single carbon source in medium， and it increased significantly after 12 h under trehalose induction （P<0.05）.In summary， Bctre1 was closely related to conidia germination and different carbon source utilization during the process of infecting host， and played an important role in promoting mycelial growth at late stage of infection.

Keywords Botrytis cinerea; Bctre1; Genome location; Protein structure; Expression analysis

灰葡萄孢（Botrytis cinerea）侵染寄主廣泛，有遗传变异大、繁殖快、侵染性强等特点。它已对多种杀菌剂产生抗药性，但农业生产中为防止灰霉病的发生，大多采用加大杀菌剂使用量的方法，这对农产品的安全性造成了严重威胁[1，2]。海藻糖是一种普遍的储藏性糖类， 有研究表明，其参与真菌的生长发育、代谢反应、逆境胁迫、产孢和致病力等途径[3-6]。海藻糖酶（trehalase，Tre）能抑制海藻糖合成，当真菌体内的海藻糖含量降低时，其生长发育与致病性均受到影响。例如，海藻糖酶能抑制假丝酵母和稻瘟病菌在寄主体内的存活[3]。黑曲霉缺失treB基因可使分生孢子发育受限和致病力下降[7]。白色念珠菌缺失treB基因可导致致病力下降[4]。Foster等[8]研究结果表明，稻瘟病菌（Magnaprothe grisea）中编码中性海藻糖酶的基因NTH1突变会引起病菌在寄主组织内扩展能力下降，导致致病力下降。……