綠僵菌胞外蛋白酶Pr1C誘導飛蝗中腸免疫功能分析

郭隆隆 李貝貝 李霜

摘要 為明確金龜子綠僵菌Metarhizium anisopliae胞外蛋白酶Pr1C在綠僵菌侵染飛蝗Locusta migratoria中腸中的作用，從綠僵菌IMI330189菌株轉錄組中獲得并分析了Pr1C基因全長序列，設計引物對該基因進行了克隆和原核表達。將表達的Pr1C蛋白與綠僵菌IMI330189混合后飼喂處理，測定了混合物對飛蝗的毒力，并通過熒光定量PCR檢測了飛蝗中腸免疫相關基因的表達情況。結果顯示，該基因屬于綠僵菌類枯草桿菌絲氨酸蛋白酶家族，全長為2 126 bp，蛋白大小為71 kDa。Pr1C與綠僵菌IMI330189混合后可以顯著提高對飛蝗的毒力。熒光定量結果表明，Defensin， Persephone， Tube， Relish， Dredd 5個基因在各處理中表達量均呈現升高趨勢，其中Pr1C蛋白和綠僵菌混合處理的變化比其他處理組變化快，于第2天達到較高水平;綠僵菌處理變化較慢，于第6天達到最高水平。本研究表明，綠僵菌胞外蛋白酶Pr1C可顯著提高綠僵菌毒力，促進綠僵菌侵染速率，為進一步研制和應用生物制劑防治飛蝗提供理論依據。

關鍵詞 綠僵菌; 胞外蛋白酶; 飛蝗; 毒力

中圖分類號： S 476.12 ?文獻標識碼： A ?DOI： 10.16688/j.zwbh.2019075

Abstract In order to explore the role of Metarhizium anisopliae in the midgut infection of Locusta migratoria， the full-length sequence of Pr1C gene was obtained from the transcriptome sequences of M. anisopliae strain IMI330189 and primers were designed to clone and express the Pr1C gene. The obtained Pr1C protein was mixed with M. anisopliae IMI330189 spore and its toxicity to the locust was checked by using bait-feeding method. The expression level of intestinal immune-related genes in locusts was detected by fluorescence quantitative PCR. NCBI Blast results showed that Pr1C gene belonged to the serine protease family of M. anisopliae with a full length of 2 126 bp， and its protein size was 71 kDa. The bioassay results showed that the mixture of Pr1C and M. anisopliae IMI330189 could significantly increase the virulence to L. migratoria. The expression level of five genes Defensin， Persephone， Tube， Relish and Dredd showed an increasing trend in all treatments， but the treatment with the mixture of Pr1C protein and metarhizium induced a faster change than other treatments and reached to the highest level on the second day. The treatment with M. anisopliae induced slow change in the expression of genes， and the highest level was observed on the sixth day. This study indicated that the extracellular protease Pr1C of M. anisopliae could significantly increase the virulence of M. anisopliae and its infection rate， providing a theoretical basis for the further development and application of biological preparations for controlling L. migratoria.

Key words Metarhizium anisopliae; extracellular protease; Locusta migratoria; virulence

金龜子綠僵菌 Metarhizium anisopliae作為一種廣泛存在的昆蟲病原真菌能夠寄生于多種害蟲，它主要通過穿透昆蟲體表進入寄主體內，消耗營養，產生毒素等方式致死害蟲[1]。Kucera在昆蟲體壁離體誘導培養條件下首次發現了綠僵菌胞外蛋白酶的活性，并指出菌株的蛋白酶活力與其毒力存在關系[2]。樊美珍等利用該方法，以馬尾松毛蟲作為試蟲，測定了球孢白僵菌胞外蛋白酶的產量與毒力的關系，進一步證實二者之間存在相關性[3]。……