杧果炭疽病菌漆酶基因Cglac3序列特征及其在两个侵染相关基因突变体中的表达分析

钟昌开 肖春丽 张贺 蒲金基 吴秋玉 刘燕莉 刘晓妹

摘  要：本研究運用同源克隆技术克隆了杧果炭疽病菌（Colletotrichum gloeosporioides）的Cglac3漆酶基因，并采用实时荧光定量PCR分析了该基因在侵染过程、漆酶基因LAC1突变体、外切葡聚糖酶基因CgCBH1突变体中的差异表达情况。结果表明，杧果炭疽病菌漆酶Cglac3基因大小为1842 bp，含3个内含子，大小分别为56、51、58 bp。Cglac3开放读码框编码558个氨基酸，蛋白分子量为61.38 kDa，等电点（PI）为4.50。与未侵染对照0 h的表达量相比，Cglac3在6 h孢子萌发形成附着胞时表达量迅速升高，在12 h侵入寄主后达到最高，之后在菌丝扩展、叶片显症过程中出现波动，但均远高于0 h的表达量，显示Cglac3在侵染的不同阶段均发挥着重要的作用，可能是胶孢炭疽菌的重要致病因子之一。与野生型相比，Cglac3表达量在杧果炭疽病菌漆酶基因LAC1敲除突变体中下降了74%，在外切葡聚糖酶基因CgCBH1敲除突变体中下降了56%;在CgCBH1缺失敲除突变体中，LAC1的表达下降了68%，与Cglac3的表现趋势一致，说明Cglac3的表达受LAC1调控，外切葡聚糖酶基因CgCBH1也会影响Cglac3和LAC1的表达。

关键词：杧果;漆酶;Cglac3基因;qRT-PCR中图分类号：S432;Q78      文献标识码：A

Sequence Characteristics of Laccase GeneCglac3 and Its Expression in Two Infection-Related Gene Mutants fromColletotrichum

gloeosporioides on Mango

ZHONG Changkai¹， XIAO Chunli¹， ZHANG He²， PU Jinji²， WU Qiuyu¹， LIU Yanli¹， LIU Xiaomei^1*

1. College of Plant Protection， Hainan University / Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests （Hainan University）， Ministry of Education， Haikou， Hainan 570228， China; 2. Environment and Plant Protection Institute， Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Integrated Pest Management on Tropical Crops， Ministry of Agriculture and Rural Affairs / Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests， Haikou， Hainan 571101， China

Abstract： In this experiment， a laccase geneCglac3was cloned  by homologously fromColletotrichumgloeosporioides， which is the fungal pathogen of mango anthracnose， and its expressions during infection process， in laccase gene Cglac1and exoglucanase gene CgCBH1deletion knockout mutants， were comparatively analyzed using real-time fluorescent quantitative PCR.Cglac3was 1842 bp in size with three introns of 56， 51 and 58 bp respectively. The open reading frame ofCglac3encoded 558 amino acids with a protein molecular weight of 61.38 kDa and an isoelectric point （PI） of 4.50.Cglac3was highly expressed at 6 h after inoculation when the fungal spores were germing and forming appressoria， increased rapidly at 12 h after inoculation when the fugus were invading. Since then， the expresson ofCglac3fluctuated during hyphal expansion and leaf necrosis， but both were much higher than the expression level at 0 h. It suggested that Cglac3played an important role duringC. gloeosporioidesinfection， might be a key virulent factor. Compared with the wild type， the expression ofCglac3decreased by 74% in theC. gloeosporioidesΔLAC1mutant， and decreased by 56% in theΔCgCBH1mutant. Interestingly， the expression ofLAC1was also decreased by 68% in theΔCgCBH1mutant， consistent with the trend ofCglac3expression， indicating thatLAC1 affected the expression ofCglac3， andCgCBH1affected both the expressions ofCglac3 andLAC1.

Keywords： mango; laccase;Cglac3gene; qRT-PCR

DOI： 10.3969/j.issn.1000-2561.2020.06.019

原产于“一带一路”沿线南亚、东南亚等国家的杧果（Mangifera indicaL.）有着“热带果王”的美誉，近年来我国杧果产业得到了长足发展，已成为热区重要的特色农业支柱产业之一，面积和产量分别位居世界第3位和第5位，但在品种选育、栽培管理、病虫害防治、采后保鲜加工等方面与上述原产国仍存在一定差距^[1]。

由胶孢炭疽菌（Colletotrichum gloeos?porioi?desPenz. & Sacc.）引起的杧果炭疽病是影响该产业健康发展的主要因素之一，该菌在细胞壁水解酶的协助下，主要借助芽管顶端产生的黑色素化的附着胞直接穿透寄主表皮侵入为害^[2]。……