摘要
【目的】为了揭示杧果胶孢炭疽病菌(Colletotrichum gloeosporioides)漆酶基因家族成员在转录水平上响应无机阴阳离子和有机物类型外源物的表达模式,为探究该家族在C.gloeosporioides侵染中的作用和开发潜在杀菌剂靶标提供参考。【方法】测定了27种外源物对杧果胶孢炭疽病菌胞外漆酶酶活性的影响,以及4种代表性外源物对胞内漆酶酶活性、黑色素产量和附着胞形成率的影响,同时利用实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)分析了4种代表性外源物对13个漆酶基因家族成员表达的调控。【结果】对杧果胶孢炭疽病菌胞外漆酶活性具有明显抑制作用的有Fe^(2+)、Fe^(3+)、NH_(4)^(+)、Na^(+)、K^(+)、SO_(3)^(2-)、CO_(3)^(2-)、HCO_(3)^(-)、SO_(4)^(2-)、NO_(3)^(-)、H_(2)PO_(4)^(-)、HPO_(4)^(2-)、半胱氨酸、白藜芦醇、对甲氧基苯胺、对羟基苯甲酸、二甲基亚砜、肉桂酸和香草酸,其中Fe^(2+)、Fe^(3+)、SO_(3)^(2-)和半胱氨酸的抑制作用为90%,尤为明显;3个浓度的Ca^(2+)均促进,Mn^(2+)、Cu^(2+)、Zn^(2+)和Mg^(2+)在1 mmol·L^(-1)时促进,5、10 mmol·L^(-1)时抑制;4-羟基-3-甲氧基肉桂酸甲酯和焦性没食子酸在1、5 mmol·L^(-1)时抑制,在10 mmol·L^(-1)时明显促进。从对胞内漆酶酶活性和附着胞形成率影响来看,1 mmol·L^(-1)的Cu^(2+)和Mn^(2+)促进,1 mmol·L^(-1)的SO_(3)^(2-)和半胱氨酸抑制。从对黑色素合成影响来看,1 mmol·L^(-1)的Cu^(2+)显著促进,但1 mmol·L^(-1)的Mn^(2+)和SO_(3)^(2-)和半胱氨酸无显著影响。qRT-PCR结果显示,1 mmol·L^(-1)的Cu^(2+)对约3/4的漆酶基因家族成员诱导表达,但抑制Cglac11和Cglac12的表达;Mn^(2+)、SO_(3)^(2-)和半胱氨酸对约1/3的成员起促进作用,对23%的成员抑制表达,不影响其余成员的调控;4种外源物均显著促进Cglac2的表达但均不影响Cglac3的表达。【结论】不同外源物对杧果胶孢炭疽病菌漆酶活性、黑色素合成和附着胞的形成以及对漆酶基因家族成员的表达调控影响有差异,初步推测Cglac2、Cglac12和Cglac13与外源物Cu^(2+)、Mn^(2+)、SO_(3)^(2-)和半胱氨酸之间存在密切关系。
【Objective】Mango(Mangifera indica Linn.)is the second most important tropical fruit in China.Colletotrichum gloeosporioides is the main pathogen of mango anthracnose,a serious disease reducing fruit production.It has large host-range and wide distributions.The main way of infection of Colletotrichum spp.is direct invasion of the melanized appressorium to the host.Melanin could maintain the integrity of the appressorium cell wall,prevent the efflux of intracellular glycerol,promote the formation of high turgor in the appressorium,and help the fungus to invade the host.So,melanin is considered to be a virulence factor for many plants pathogenic fungi.The intracellular melanin of Colletotrichum is mostly dihydroxynaphthalene(DHN)melanin,whose synthesis pathway is composed of a number of key enzymes with the laccase,the last enzyme,which plays a role in oxidative polymerization and catalyzes the synthesis of melanin.The laccase is a polyphenol oxidase containing copper ions,and widely distributed in plants,fungi,bacteria,lichens and insects.The laccases often exist in the form of gene superfamily and are considered as potential drug targets.In addition to being influenced by culture conditions,the expression of laccase genes is also regulated by a variety of exogenous substances such as metal cations,inorganic anions and organic substances,in a dose-response manner.In previous work,we cloned 13 laccase genes from C.gloeosporioides.In order to know the expression pattern of the laccase gene family,their roles in pathogenesis of C.gloeosporioides,and subsequently explore their possibility as potential target of fungicides or adjuvants,the influence of exogenous substances on the laccase activity,melanin production,appressorium formation and the expression of 13 laccase genes were analyzed in this paper.【Methods】The crude laccase was obtained by filtration and centrifugation from C.gloeosporioides cultures when the extracellular laccase activity reached the peak.The effects of 18 anions and cations and 9 organic substances on the extracellular laccase activity were tested using a laccase kit.On this basis,4 representative exogenous substances were selected and their effects on the internal laccase activity,melanin production and appressorium formation rate were determined.The expression of 13 laccase genes after treatment of 4 representative exogenous substances was analyzed by qRT-PCR,respectively.【Results】The extracellular laccase secretion of C.gloeosporioides reached the peak10 days after being cultuted in liquid medium.Among 27 tested exogenous substances,20 showed inhibitory effects on the extracellular laccase activity,including Fe^(2+),Fe^(3+),NH_(4)^(+),Na^(+),K^(+),Cl^(-),SO_(3)^(2-),CO_(3)^(2-),HCO_(3)^(-),SO_(4)^(2-),NO_(3)^(-),H_(2)PO_(4)^(-),HPO_(4)^(2-),cysteine,resveratrol,p-aniline,p-hydroxybenzoic acid,dimethyl sulfoxide,cinnamic acid and vanillic acid.The inhibitory effect of Fe^(2+),Fe^(3+),SO_(3)^(2-) and cysteine was particularly obvious,with an inhibition rate over 90%.Ca^(2+)at 1,5 and 10 mmol·L^(-1)all promoted laccase activity,while Mn^(2+),Cu^(2+),Zn^(2+)and Mg^(2+)promoted at 1 mmol·L^(-1),and inhibited at 5 and 10 mmol·L^(-1).Methyl 4-hydroxy-3-methoxycinnamate and pyrogallonic acid inhibited at 1 and 5 mmol·L^(-1),but significantly promoted at 10 mmol·L^(-1).For the representative exogenous substances,1 mmol·L^(-1)Cu^(2+)and Mn^(2+)promoted intracellular laccase activity and appressorium formation,and promotion of Cu^(2+)was more obviously than Mn^(2+),with the relative laccase activity of 149%.1 mmol·L^(-1)SO_(3)^(2-) and cysteine inhibited intracellular laccase activity and appressorium formation,with the relative laccase activity of 74%and 31%,respectively.For melanin synthesis,1 mmol·L^(-1)Cu^(2+)significantly promoted melanin production,the melanin content increased by 11.36%compared with the control,while Mn^(2+),SO_(3)^(2-)and cysteine had no significant effect.The qRT-PCR results showed that 1 mmol·L^(-1)Cu^(2+)induced the expression of about 3/4 of laccase genes,but inhibited the expression of the Cglac11 and Cglac12,and had no effect on the expression of the Cglac3.Mn^(2+),SO_(3)^(2-) and cysteine promoted the expression of about 1/3 of laccase genes,inhibited the expression of about 23%of laccase genes,and did not affect the expression of the rest laccase genes.Four exogenous substances significantly promoted the expression of the Cglac2,and had no effect on the expression of Cglac3【.Conclusions】The different exogenous substances at different concentrations showed different effects on the extracellular laccase activity of C.gloeosporioides.Cu^(2+),Mn^(2+),SO_(3)^(2-)and cysteine had different effects on intracellular enzyme activity,appressorium formation and melanin production,as well as the expression of 13 laccase genes.It was preliminarily speculated that laccase genes Cglac2,Cglac12 and Cglac13 would be closely related to the exogenous Cu^(2+),Mn^(2+),SO_(3)^(2-)and cysteine.
作者
董玲玲
谭晴
张梦婷
肖春丽
张贺
蒲金基
刘晓妹
DONG Lingling;TAN Qing;ZHANG Mengting;XIAO Chunli;ZHANG He;PU Jinji;LIU Xiaomei(College of Plant Protection/Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests,Ministry of Education,Hainan University,Haikou 570228,Hainan,China;Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Integrated Pest Management of Tropical Crops,Ministry of Agriculture and Rural Affairs,Haikou 571101,Hainan,China)
出处
《果树学报》
CAS
CSCD
北大核心
2022年第7期1157-1166,共10页
Journal of Fruit Science
基金
国家自然科学基金项目(31860479,31460455)。
关键词
杧果胶孢炭疽病菌
漆酶基因家族
离子
有机物
Colletotrichum gloeosporioides
Laccase gene family
Ion
Organic substances
作者简介
董玲玲,女,在读硕士研究生,研究方向为植物病理学。Tel:18889802387,E-mail:1620465865@qq.com;通信作者:蒲金基.E-mail:simmonpjj@163.com;通信作者:刘晓妹.E-mail:E-mail:lxmpll@126.com。
