红火蚁Toll受体家族免疫响应绿僵菌表达模式的研究_许亚婷.pdf

环境昆虫学报2022，44(6):1528 1540http:Journal of Environmental Entomologydoi:10.3969/j.issn.1674 0858.2022.06.23许亚婷，杨荣蓉，吴洪鑫，陆永跃，金丰良，许小霞 红火蚁 Toll 受体家族免疫响应绿僵菌表达模式的研究 J 环境昆虫学报，2022，44(6):1528 1540红火蚁 Toll 受体家族免疫响应绿僵菌表达模式的研究许亚婷，杨荣蓉，吴洪鑫，陆永跃，金丰良，许小霞*(华南农业大学植物保护学院昆虫学系，广东省生物农药创制与应用重点实验室，“一带一路”农业有害生物绿色防控科技产业创新院，广州 510642)摘要:为探究红火蚁 Solenopsis invicta Buren Toll 受体家族基因如何免疫响应绿僵菌 Metarhizium 的侵染。本研究采用浸渍法测定了不同浓度下绿僵菌对红火蚁大中小 3 种不同大小工蚁的致病力，并用显微镜观察了绿僵菌侵染后红火蚁 3 种工蚁的表型变化。利用生物信息学筛选 Toll 受体基因，并对 Toll 受体家族基因的理化性质、结构域、染色体位置、系统进化进行分析鉴定。运用实时荧光定量 PC(T-qPC)检测了红火蚁大中小型工蚁中Toll 受体家族的发育历期和绿僵菌侵染后的表达模式。结果表明，绿僵菌在 96 h 对红火蚁大中小型工蚁的致死中浓度(LC50)分别为 5.8 107孢子/mL、3.1 107孢子/mL、1.5 107孢子/mL;绿僵菌侵染红火蚁第 3 天，显微镜下观察到红火蚁体壁细小的菌丝，虫体僵硬，寄主死亡后菌丝迅速蔓延并逐渐长成橄榄绿色分生孢子;T-qPC 结果表明 Toll 受体基因在红火蚁不同发育阶段中的 mNA 表达水平存在显著差异，且 Toll 受体基因在雌性生殖蚁表达水平显著高于雄性生殖蚁;T-qPC 结果表明红火蚁大中小型工蚁 Toll 受体免疫响应绿僵菌不一样。在大型工蚁中，绿僵菌侵染后，Toll 受体家族基因能显著上调表达，6 h 是一个诱导高峰期，Toll2-1 和L 转录水平最高，响应绿僵菌最强;在中型工蚁中，绿僵菌不能刺激 Toll2-2 基因的表达，却能强烈诱导Toll1，Toll2-1，Toll6，Toll7 和 L 基因的上调表达，Toll1 和 Toll2-1 响应绿僵菌最强。在小型工蚁中，绿僵菌能显著诱导 Toll 受体家族基因基因的上调表达，在 24 h 时，L 基因表达量最高，相比于对照，L 基因表达提高 25 倍，L 在 6 h 和 24 h 响应绿僵菌最强。以上研究表明 Toll 受体可以免疫响应入侵的绿僵菌，且不同的Toll 对绿僵菌可能具有不同的响应机制。本研究为进一步阐明 Toll 受体的功能奠定基础，为进一步利用绿僵菌控制红火蚁提供技术指导。关键词:红火蚁;Toll;绿僵菌;表达模式中图分类号:Q968.1;S433;S476文献标识码:A文章编号:1674 0858(2022)06 1528 13基金项目:国家重点研发计划项目(2021YFD1000500)作者简介:许亚婷，女，1997 年生，江西吉安人，硕士研究生，研究方向为农业昆虫与害虫防治，E mail:xuyt1101 *通讯作者 Author for correspondence:许小霞，女，博士，副教授，研究方向为生物防治，E mail:xuxiaoxia111 收稿日期 eceived:2022 09 08;接受日期 Accepted:2022 10 15Study on expression pattern of Toll receptor family of Solenopsis invictaBuren in response to Metarhizium anisopliaeXU Ya-Ting，YANG ong-ong，WU Hong-Xin，LU Yong-Yue，JIN Feng-Liang，XU Xiao-Xia*(Key Laboratory of Bio-Pesticide Creation and Application of Guangdong Province，Department ofEntomology，College of Plant Protection，South China Agricultural University，Guangzhou 510642，China)Abstract:To investigate the immune response of Toll receptor family gene of Solenopsis invicta toMetarhizium anisopliae infection.In this study，the impregnation method was used to determine the6 期许亚婷等:红火蚁 Toll 受体家族免疫响应绿僵菌表达模式的研究pathogenicity of M.anisopliae infection three different grades of worker S.invicta，and the phenotypicchanges of S.invicta after M.anisopliae infection were photographed with the microscope.Based on thegenome of S.invicta，the Toll receptor gene family sequence were screened by bioinformatics analysis，the physical and the chemical properties，structural domain，chromosomal location and phylogeneticevolution of the Toll receptor family gene were analyzed.eal time quantitative PC(T-qPC)wasused to analyze the expression patterns of the Toll receptor family of developmental stage in major，medium and minor-sized worker ants and the induced expression patterns of the Toll receptor family inants infected by M.anisopliae.The results showed that the lethal medium concentration(LC50)at 96 hof M.anisopliae in major，medium and minor-sized worker ants.LC50were 5.8 107spores/mL，3.1 107spores/mL and 1.5 107spores/mL，respectively.On the third day of M.anisopliae infection，small mycelia could be observed on the body of S.invicta.The mycelia spread rapidly and gradually grewinto olive green conidia after the death of the host.The results of T-qPC indicated that the mNAlevel of Toll receptor gene was significantly different in developmental stages of S.invicta，especially，theexpression level of Toll receptor family in female were much higher than in male S.Invicta.The T-qPC results also showed that the immune response of Toll receptors to M.anisopliae was different.Inmajor-sized worker ants，the expression level of Toll receptor family genes were significantly upregulatedafter M.anisopliae infection，and the induction peak was 6 h.The transcription levels of Toll2-1 andL were the highest，and the response to M.anisopliae was the strongest.In medium-sized workerants，M.anisopliae could not stimulate the expression of Toll2-2 gene，but could strongly induce the up-regulated expression of Toll1，Toll2-1，Toll6，Toll7 and L genes，and Toll1 and Toll2-1 were the mostresponsive to M.anisopliae.In the minor-sized worker ants，M.anisopliae could significantly induce theup-regulated expression of Toll receptor family genes.The L gene was the highest expression level with25 times higher than the control at 24 h post infection.The L was the most responsive to M.anisopliaeat 6 h and 24 h.These studies indicated that Toll receptors could be immune to invading M.anisopliae，and different Toll might have different mechanism response against M.anisopliae.This study laid thefoundation for further elucidate the function of Toll receptor and provided technical guidance for furthercontrol of S.invicta by M.anisopliaeKey words:S.invicta;M.anisopliae;Toll;expression patterns红火蚁 Solenopsis invicta Buren 属于膜翅目蚁科，原分布于南美洲巴拉那河流域，2003 年侵入我国台湾桃园与嘉义地区，2004 年传入中国广东省吴川市。红火蚁具有强大的侵略性、攻击性、适应性、繁殖快、竞争力强、食性杂的特点。红火蚁对人体健康、公共安全、农林业生产和生态环境均具有严重的危害性，是世界公认的最具危险的外来入侵物种之一。红火蚁在我国基本上是多蚁后种群，广东省光温条件特别适合其繁殖，且红火蚁在我国尚无天敌，能快速形成高密度种群(刘 晓 燕