飛蝗幾丁質(zhì)脫乙�；富虻姆肿犹匦院蜕飳W(xué)功能
發(fā)布時(shí)間：2018-10-05 15:14
【摘要】：【目的】幾丁質(zhì)脫乙酰基酶(chitin deacetylase,CDA)是昆蟲幾丁質(zhì)代謝系統(tǒng)的重要酶系,研究飛蝗(Locusta migratoria)幾丁質(zhì)脫乙�；富虻姆肿犹匦院蜕飳W(xué)功能,為新型農(nóng)藥靶標(biāo)篩選提供科學(xué)依據(jù)�！痉椒ā炕陲w蝗轉(zhuǎn)錄組數(shù)據(jù)庫(kù),獲得幾丁質(zhì)脫乙�；富虻腸 DNA序列,將其與飛蝗基因組進(jìn)行比對(duì),繪制基因結(jié)構(gòu)圖。將其與赤擬谷盜CDAs序列進(jìn)行比對(duì),并采用Blast P和SMART軟件進(jìn)行功能域預(yù)測(cè)。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(jìn)行聚類分析,采用MEGA 5.02軟件中的neighbor-joining(NJ)法構(gòu)建系統(tǒng)發(fā)育樹。采用reverse transcription quantitative PCR(RT-q PCR)方法檢測(cè)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(dá)特性,進(jìn)一步采用RNA干擾(RNAi)技術(shù)研究其對(duì)飛蝗蛻皮發(fā)育的影響。采用化學(xué)檢測(cè)法測(cè)定其幾丁質(zhì)含量。利用透射電鏡(TEM)觀察其對(duì)表皮幾丁質(zhì)排列的影響。【結(jié)果】在飛蝗轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中搜索獲得3條幾丁質(zhì)脫乙�；富蛉L(zhǎng)c DNA序列,生物信息學(xué)分析發(fā)現(xiàn)其均具有信號(hào)肽,開放閱讀框包含幾丁質(zhì)結(jié)合區(qū)(Ch BD)和幾丁質(zhì)脫乙�；呋�(CDA)2個(gè)功能域。與赤擬谷盜CDAs序列比對(duì)結(jié)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(gè)剪切子差異序列位置一致,顯示2條序列為2個(gè)剪切子。聚類分析結(jié)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L(zhǎng)m CDA4、Lm CDA5a和Lm CDA5b。不同組織部位表達(dá)結(jié)果表明Lm CDA4和Lm CDA5在飛蝗前腸、后腸和表皮中高表達(dá),Lm CDA5在脂肪體中也有較高的表達(dá);CDAs在5齡不同天數(shù)表皮表達(dá)結(jié)果顯示Lm CDA4和Lm CDA5在5齡第1和第2天的表皮中表達(dá)量較高,之后逐步下降。注射ds Lm CDA4或ds Lm CDA524 h后,與對(duì)照組相比,基因表達(dá)量顯著降低,沉默效率達(dá)到98.1%和95.6%,但均無(wú)可見表型,都可正常蛻皮至成蟲。幾丁質(zhì)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(zhì)含量及排列�！窘Y(jié)論】飛蝗幾丁質(zhì)脫乙酰基酶4和5基因主要在飛蝗表皮和前/后腸表達(dá),生物學(xué)功能研究表明這2個(gè)基因不參與飛蝗蛻皮發(fā)育過(guò)程,靶基因沉默對(duì)飛蝗生長(zhǎng)發(fā)育及表皮結(jié)構(gòu)無(wú)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(xué)應(yīng)用生物學(xué)研究所;山西大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(31672364) 山西省基礎(chǔ)研究計(jì)劃(2015011070) 山西省回國(guó)留學(xué)人員科研資助項(xiàng)目(2015-007) 山西省高等學(xué)�？萍紕�(chuàng)新項(xiàng)目(2017104) 山西省研究生優(yōu)秀創(chuàng)新項(xiàng)目(02180114092041)
【分類號(hào)】：S433.2
本文編號(hào)：2253866
[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(xué)應(yīng)用生物學(xué)研究所;山西大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(31672364) 山西省基礎(chǔ)研究計(jì)劃(2015011070) 山西省回國(guó)留學(xué)人員科研資助項(xiàng)目(2015-007) 山西省高等學(xué)�？萍紕�(chuàng)新項(xiàng)目(2017104) 山西省研究生優(yōu)秀創(chuàng)新項(xiàng)目(02180114092041)
【分類號(hào)】：S433.2
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