九香蟲i型溶菌酶基因的克隆與細菌誘導表達分析

黃海 杜娟 李尚偉 龔濤 齊小浪

摘要

溶菌酶是一組進化上保守的酶，在昆蟲中主要分為c型和i型兩類。為明確i型溶菌酶在九香蟲Coridius chinensis體內的表達模式與功能，我們對九香蟲的兩個i型溶菌酶基因CcLysi1和CcLysi2進行了克隆，這兩個基因的編碼區分別為504 bp和432 bp，分別編碼167和143個氨基酸。序列分析顯示，CcLysi1和CcLysi2缺乏與催化活性相關的谷氨酸（E）和絲氨酸（S）。同源性和聚類分析顯示，CcLysi1和CcLysi2與斯氏珀蝽Plautia stali的i型溶菌酶相似性最高，分別為80.36%和48.95%。采用實時熒光定量PCR（RTqPCR）解析CcLysi1和CcLysi2基因的時空表達譜，結果表明它們在九香蟲不同發育階段都有表達，CcLysi1在成蟲中表達水平最高，而CcLysi2在5齡若蟲中表達水平最高;它們在所檢測的成蟲不同組織中都有表達，都在脂肪體中表達水平最高。九香蟲被注射細菌后24 h，CcLysi1和CcLysi2的表達顯著上調;九香蟲被飼喂細菌后，這兩種基因的表達水平沒有顯著變化。九香蟲的這兩種i型溶菌酶基因的表達受到自身免疫機制的調控，可以被誘導而參與免疫應答，但不具有參與消化的功能。該研究為進一步明確九香蟲i型溶菌酶基因的功能奠定基礎。

??關鍵詞

九香蟲;i型溶菌酶;時空表達;先天免疫;細菌誘導

中圖分類號：Q967

文獻標識碼：A

DOI：10.16688/j.zwbh.2020669

Cloning and induced expression analysis of the itype lysozyme genes in Coridius chinensis

HUANG Hai，DU Juan，LI Shangwei*，GONG Tao，QI Xiaolang

（Guizhou Provincial Key Laboratory for Agricultural Pest Management in Mountainous Regions，

Institute of Entomology， Guizhou University， Guiyang， Guizhou550025， China）

Abstract

Lysozymes are a group of evolutionarily conserved enzymes and can be divided into ctype and itype in insects. In order to clarify the expression pattern and function of the itype lysozyme in Coridius chinensis， we cloned two itype lysozyme genes CcLysi1 and CcLysi2. The coding regions of these two genes were 504 bp and 432 bp， encoding 167 and 143 amino acids， respectively. Sequence analysis showed that CcLysi1 and CcLysi2 lacked glutamic acid （E） and serine （S） related to catalytic activity. Homology and cluster analyses showed that CcLysi1 and CcLysi2

shared the similarity of 80.36% and 48.95%， respectively， with the itype lysozyme from Plautia stali. The spatiotemporal expression profiles of CcLysi1 and CcLysi2 were analyzed by using realtime quantitative PCR （RTqPCR）. The results indicated that these two genes were expressed at various developmental stages of C. chinensis， with the highest expression levels for CcLysi1 in the adults and for CcLysi2 in the fifthinstar nymphs; they were expressed in different tissues of adults and had the highest expression level in the fat bodies. The expression levels of CcLysi1 and CcLysi2 were significantly upregulated within 24 h post bacterial injection， but there were no significant changes in the expression levels of these two genes post bacterial feeding. The expression of these two itype lysozyme genes in C.chinensis was regulated by its own immune mechanism and could be induced to participate in immune response， but they were not involved in digestion. This study laid a foundation for further elucidating the functions of the itype lysozyme genes in C.chinensis.

Key words

Coridius chinensis;itype lysozyme;spatiotemporal expression;innate immunity;bacterial induction

九香蟲Coridius chinensis隸屬于半翅目Hemiptera兜蝽科Dinidoridae，廣泛分布于我國南方地區，包括云南、四川、貴州、廣西等地，主要以葫蘆科植物的汁液為食，春夏季節常棲息在南瓜等農作物的莖葉上吸食漿液，造成植株生長緩慢，為害嚴重時，可造成植株枯死。 同時，九香蟲也是一種極具開發潛力的昆蟲資源，其蟲體內富含蛋白質、多肽、脂肪酸、維生素以及微量元素等[1- 2]，具有很高的食用和藥用價值[3]?！?br>