飞蝗几丁质脱乙酰基酶基因的分子特性和生物学功能
发布时间：2018-10-05 15:14
【摘要】：【目的】几丁质脱乙酰基酶(chitin deacetylase,CDA)是昆虫几丁质代谢系统的重要酶系,研究飞蝗(Locusta migratoria)几丁质脱乙酰基酶基因的分子特性和生物学功能,为新型农药靶标筛选提供科学依据。【方法】基于飞蝗转录组数据库,获得几丁质脱乙酰基酶基因的c DNA序列,将其与飞蝗基因组进行比对,绘制基因结构图。将其与赤拟谷盗CDAs序列进行比对,并采用Blast P和SMART软件进行功能域预测。将已鉴定的飞蝗CDAs分别与赤拟谷盗、果蝇、冈比亚按蚊、家蚕、中华稻蝗和云杉卷叶蛾的同源序列进行聚类分析,采用MEGA 5.02软件中的neighbor-joining(NJ)法构建系统发育树。采用reverse transcription quantitative PCR(RT-q PCR)方法检测分析CDA在飞蝗5龄若虫不同组织部位和不同发育日龄表皮中的表达特性,进一步采用RNA干扰(RNAi)技术研究其对飞蝗蜕皮发育的影响。采用化学检测法测定其几丁质含量。利用透射电镜(TEM)观察其对表皮几丁质排列的影响。【结果】在飞蝗转录组数据库中搜索获得3条几丁质脱乙酰基酶基因全长c DNA序列,生物信息学分析发现其均具有信号肽,开放阅读框包含几丁质结合区(Ch BD)和几丁质脱乙酰基催化域(CDA)2个功能域。与赤拟谷盗CDAs序列比对结果表明飞蝗2条CDAs部分序列存在差异,且与赤拟谷盗Tc CDA5的2个剪切子差异序列位置一致,显示2条序列为2个剪切子。聚类分析结果表明,3条序列分别与6种昆虫CDA4和CDA5以较高的置信度聚为一支。分别将其命名为Lm CDA4、Lm CDA5a和Lm CDA5b。不同组织部位表达结果表明Lm CDA4和Lm CDA5在飞蝗前肠、后肠和表皮中高表达,Lm CDA5在脂肪体中也有较高的表达;CDAs在5龄不同天数表皮表达结果显示Lm CDA4和Lm CDA5在5龄第1和第2天的表皮中表达量较高,之后逐步下降。注射ds Lm CDA4或ds Lm CDA524 h后,与对照组相比,基因表达量显著降低,沉默效率达到98.1%和95.6%,但均无可见表型,都可正常蜕皮至成虫。几丁质含量和透射电镜分析显示,上述基因RNAi后不影响表皮几丁质含量及排列。【结论】飞蝗几丁质脱乙酰基酶4和5基因主要在飞蝗表皮和前/后肠表达,生物学功能研究表明这2个基因不参与飞蝗蜕皮发育过程,靶基因沉默对飞蝗生长发育及表皮结构无影响。
[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.
【作者单位】： 山西大学应用生物学研究所;山西大学生命科学学院;
【基金】：国家自然科学基金(31672364) 山西省基础研究计划(2015011070) 山西省回国留学人员科研资助项目(2015-007) 山西省高等学校科技创新项目(2017104) 山西省研究生优秀创新项目(02180114092041)
【分类号】：S433.2
本文编号：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.
【作者单位】： 山西大学应用生物学研究所;山西大学生命科学学院;
【基金】：国家自然科学基金(31672364) 山西省基础研究计划(2015011070) 山西省回国留学人员科研资助项目(2015-007) 山西省高等学校科技创新项目(2017104) 山西省研究生优秀创新项目(02180114092041)
【分类号】：S433.2
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