【摘要】：【目的】幾丁質脫乙�；�(chitin deacetylase,CDA)是昆蟲幾丁質代謝系統(tǒng)的重要酶系,研究飛蝗(Locusta migratoria)幾丁質脫乙�；富虻姆肿犹匦院蜕飳W功能,為新型農藥靶標篩選提供科學依據�！痉椒ā炕陲w蝗轉錄組數據庫,獲得幾丁質脫乙酰基酶基因的c DNA序列,將其與飛蝗基因組進行比對,繪制基因結構圖。將其與赤擬谷盜CDAs序列進行比對,并采用Blast P和SMART軟件進行功能域預測。將已鑒定的飛蝗CDAs分別與赤擬谷盜、果蠅、岡比亞按蚊、家蠶、中華稻蝗和云杉卷葉蛾的同源序列進行聚類分析,采用MEGA 5.02軟件中的neighbor-joining(NJ)法構建系統(tǒng)發(fā)育樹。采用reverse transcription quantitative PCR(RT-q PCR)方法檢測分析CDA在飛蝗5齡若蟲不同組織部位和不同發(fā)育日齡表皮中的表達特性,進一步采用RNA干擾(RNAi)技術研究其對飛蝗蛻皮發(fā)育的影響。采用化學檢測法測定其幾丁質含量。利用透射電鏡(TEM)觀察其對表皮幾丁質排列的影響。【結果】在飛蝗轉錄組數據庫中搜索獲得3條幾丁質脫乙�；富蛉Lc DNA序列,生物信息學分析發(fā)現(xiàn)其均具有信號肽,開放閱讀框包含幾丁質結合區(qū)(Ch BD)和幾丁質脫乙酰基催化域(CDA)2個功能域。與赤擬谷盜CDAs序列比對結果表明飛蝗2條CDAs部分序列存在差異,且與赤擬谷盜Tc CDA5的2個剪切子差異序列位置一致,顯示2條序列為2個剪切子。聚類分析結果表明,3條序列分別與6種昆蟲CDA4和CDA5以較高的置信度聚為一支。分別將其命名為Lm CDA4、Lm CDA5a和Lm CDA5b。不同組織部位表達結果表明Lm CDA4和Lm CDA5在飛蝗前腸、后腸和表皮中高表達,Lm CDA5在脂肪體中也有較高的表達;CDAs在5齡不同天數表皮表達結果顯示Lm CDA4和Lm CDA5在5齡第1和第2天的表皮中表達量較高,之后逐步下降。注射ds Lm CDA4或ds Lm CDA524 h后,與對照組相比,基因表達量顯著降低,沉默效率達到98.1%和95.6%,但均無可見表型,都可正常蛻皮至成蟲。幾丁質含量和透射電鏡分析顯示,上述基因RNAi后不影響表皮幾丁質含量及排列�！窘Y論】飛蝗幾丁質脫乙�；�4和5基因主要在飛蝗表皮和前/后腸表達,生物學功能研究表明這2個基因不參與飛蝗蛻皮發(fā)育過程,靶基因沉默對飛蝗生長發(fā)育及表皮結構無影響。
[Abstract]:[objective] chitin deacetylase (chitin deacetylase,CDA) is an important enzyme system in insect chitin metabolism system. The molecular characteristics and biological functions of (Locusta migratoria) chitinase gene were studied. [methods] the c DNA sequence of chitin deacetylase gene was obtained based on the transcriptional database of locust migratory locust. It was compared with CDAs sequence and predicted by Blast P and SMART software. The identified locust CDAs was clustered with the homologous sequences of Anopheles gambiae, Bombyx mori, Anopheles gambiae, and spruce leaf moth respectively. Phylogenetic tree was constructed by neighbor-joining (NJ) method in MEGA 5.02. Reverse transcription quantitative PCR (RT-q PCR was used to detect and analyze the expression of CDA in different tissues and epidermis of fifth instar nymphs, and RNA interference (RNAi) technique was used to study the effect of CDA on decidua development of migratory locust. The chitin content was determined by chemical detection. Transmission electron microscope (TEM) was used to observe its effect on the arrangement of chitin in epidermis. [results] three full-length c DNA sequences of chitinase gene were obtained by searching the database of locust transcriptome. Bioinformatics analysis showed that all of them had signal peptides. The open reading frame contains two functional domains: chitin binding region (Ch BD) and chitin deacetyl catalytic domain (CDA). The results of alignment with the CDAs sequence showed that there were differences between the two partial sequences of CDAs, and they were in the same position as the two shears of Tc CDA5, which showed that the two sequences were two shears. The results of cluster analysis showed that the three sequences were clustered into one branch with high confidence with CDA4 and CDA5 of 6 species of insects respectively. Name it Lm CDA4,Lm CDA5a and Lm CDA5b., respectively The expression of Lm CDA4 and Lm CDA5 in the foregut of locust migratory locust showed that The high expression of Lm CDA5 was also found in the fat body. The results showed that the expression of Lm CDA4 and Lm CDA5 in the epidermis of the first and second days of the 5th instar was higher than that in the first and second day of the 5th instar, and then decreased gradually. Compared with the control group, the gene expression level decreased significantly after ds Lm CDA4 or ds Lm CDA524 injection, the silencing efficiency reached 98.1% and 95.6%, but no phenotype was observed. Chitin content and transmission electron microscope analysis showed that the above genes had no effect on the content and arrangement of epidermal chitin after RNAi. [conclusion] chitin deacetylase 4 and 5 genes were mainly expressed in the epidermis and anterior / hindgut of locust migratory locust. The biological function study showed that these two genes were not involved in the ecdylastic development of migratory locust, and the target gene silencing had no effect on the growth and epidermal structure of migratory locust.
【作者單位】： 山西大學應用生物學研究所;山西大學生命科學學院;
【基金】：國家自然科學基金(31672364) 山西省基礎研究計劃(2015011070) 山西省回國留學人員科研資助項目(2015-007) 山西省高等學�？萍紕�(chuàng)新項目(2017104) 山西省研究生優(yōu)秀創(chuàng)新項目(02180114092041)
【分類號】：S433.2

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