飞蝗β-N-乙酰氨基葡萄糖苷酶基因的克隆、表达及功能研究
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摘要
β-N-乙酰氨基葡萄糖苷酶(β-N-acetylglucosaminidase,NAG)广泛分布于细菌、真菌和节肢动物内。昆虫β-N-乙酰氨基葡萄糖苷酶通常由多个基因编码,分别或共同参与几丁质降解、糖聚合物降解及N-糖基化修饰等生理过程。由于几丁质是昆虫不可或缺的组成成分,而其在高等动植物中没有分布,故对参与几丁质降解的飞蝗β-N-乙酰氨基葡萄糖苷酶的研究可为飞蝗的防治提供新的靶标和思路。本文的主要研究内容包括：
一、飞蝗β-N-乙酰氨基葡萄糖苷酶基因全长序列的克隆、序列分析及系统进化发育树的构建。搜索飞蝗EST数据库获得ETS序列,经分析为基因的全长序列,据此设计引物,以飞蝗五龄整虫为模板扩增飞蝗β-N-乙酰氨基葡萄糖苷酶基因的全长序列,获得cDNA序列2667bp,其中开放阅读框1845bp,编码614个氨基酸,5'非翻译区(5'-UTR)和3'-非翻译区(3'-UTR)的长度分别为233bp和589bp。构建系统进化树表明该基因属于具有几丁质催化活性的NAG1家族。
二、飞蝗β-N-乙酰氨基葡萄糖苷酶基因LmNAG1的时空表达。运用RT-PCR和Real-time PCR技术对飞蝗β-N-乙酰氨基葡萄糖苷酶基因在不同龄期、五龄5、6、7天六个不同组织部位、四龄到五龄表皮中的表达进行了分析。结果显示该基因在飞蝗各个龄期均有表达,表达量随龄期递增；不同组织部位分析显示该基因在前后肠表达量最高；四龄和五龄表皮中的表达变化与20-E滴度吻合,在蜕皮前两天表达量最高。
三、飞蝗β-N-乙酰氨基葡萄糖苷酶基因LmNAG1的生物学功能研究。对LmNAG1进行RNA干扰实验,结果表明：二龄和五龄飞蝗若虫注射dsLmNAG1后,目的基因被沉默,在各组织部位的表达量均显著降低,沉默持续时间>72h；注射后的若虫表现为蜕皮时间延迟、新旧表皮无法完全分离而死亡
四、飞蝗β-N-乙酰氨基葡萄糖苷酶总酶活的测定分析。对五龄5、6和7天飞蝗不同组织部位及整虫酶活性进行测定;RNA干扰后不同时间点测定β-N-乙酰氨基葡萄糖苷酶总酶活性。结果显示：九龄最后3天该酶活性与其生理过程相关；已获得的LmNAG1基因被沉默后,β-N-乙酰氨基葡萄糖苷酶总酶活性显著下降。
综上所述,本文获得的飞蝗β-N-乙酰氨基葡萄糖苷酶基因在飞蝗生长发育过程中具有重要作用,其生物学功能与飞蝗蜕皮这一生理过程密切相关,该基因的沉默可导致飞蝗因无法完成蜕皮而死亡,且对β-N-乙酰氨基葡萄糖苷酶总酶活性具有显著的影响。该基因对于RNAi介导的飞蝗有效防治具有潜在的应用价值。
β-N-acetylglucosaminidases are widespread in bacteria, fungi and arthropods. There are often multiple genes that encoding P-N-acetylglucosaminidases in various insect species, which involve in different physiological processes including chitin degradation, glycoconjugates degradation and N-glycan modification. Because chitin is an indispensable structural component for insects but absent in higher plants and animals, the β-N-acetylglucosaminidases which is involved in chitin degradation could be an ideal potential target for pest control. The main contents of this thesis are as follows:
1. Cloning, sequencing and phylogenetic analysis of β-N-acetylglucosaminidase in Locusta migratoria.17β-N-acetylglucosaminidase gene fragments from L. migratoria were identified by searching the sequences in the EST database. Fifth-instar nymphs were used for template to obtain full-length sequence of putative LmNAGl. The full-length cDNA consists of2667nucleotides, including an open reading frame (ORF) of1845nucleotides that encode614amino acid residues and233-and589-bp non-coding regions at the5'-and3'-end of the cDNA, respectively. Phylogenetic analysis of the NAGs from L. migratoria and several other insect species showed that LmNAGl was grouped with the enzymatically characterized NAGs in group I.
2. Developmental stage and tissue-specific expression patterns of LmNAGl. RT-PCR and Real-time PCR were carried out to analyze the expression patterns of LmNAGl. Six selected tissues were dissected from day5,6and7of fifth-instar nymphs for tissue-specific expression analysis. The whole insects from the third day of each stage and integument dissected from each day during4th and5th-instar nymph stages of L. migratoria were used for developmental expression analysis. The results showed that LmNAGl was expressed in all the developmental stages and increased along with insect growth. The qPCR analyses showed that LmNAGl was predominantly expressed in foregut and hindgut, relatively low in the integument, midgut and wing pad, but hardly detectable in gastric caeca. The expression of LmNAGl in integument could be detected in whole4th-and5th-instar nymphs. The expression level of LmNAGl was significant high in the last two days of each instar, which is accordant with the ecdysone hormone (20-E) titer peak in both4th-and5th-instar nymphs.
3. Biofunctional analysis of LmNAGl gene. RNAi experiment was carried out to determine the biological function of LmNAGl. The results showed target gene could be down-regulated effectively in all selected tissues after dsLmNAGl injection, and the silence time could be kept more than72hours. The abnormal nymphs showed an "incomplete" ecdysis phenotype after the dsRNA injection and most of them eventually died from failed molting.
4. β-N-acetylglucosaminidase total activity assay. The experiment determined the β-N-acetylglucosaminidase total activity of six selected tissues from day5,6and7of L. migratoria5th-instar nymphs. The results showed that the distribution of the enzyme activity obviously change in the six tissues of the last three days of5th-instar nymphs and the patterns correspond with the process of molting and other crucial physiological functions of insect. The total activity of LmNAG starts to significantly decrease between24h and48h after LmNAGl dsRNA injection.
To summarize, the obtained LmNAGl gene plays a significant role in the developmental process of L. migratoria. Its biological function is closely related with the important process of molting. The knock-down of the gene could cause the mortality of the insect because of incomplete ecdysis and also supress suppress the total enzyme activity of β-N-acetylglucosaminidase in L. migratoria. The results suggest LmNAGl is a potential and promising target for RNAi-mediated biological pest control.
一、飞蝗β-N-乙酰氨基葡萄糖苷酶基因全长序列的克隆、序列分析及系统进化发育树的构建。搜索飞蝗EST数据库获得ETS序列,经分析为基因的全长序列,据此设计引物,以飞蝗五龄整虫为模板扩增飞蝗β-N-乙酰氨基葡萄糖苷酶基因的全长序列,获得cDNA序列2667bp,其中开放阅读框1845bp,编码614个氨基酸,5'非翻译区(5'-UTR)和3'-非翻译区(3'-UTR)的长度分别为233bp和589bp。构建系统进化树表明该基因属于具有几丁质催化活性的NAG1家族。
二、飞蝗β-N-乙酰氨基葡萄糖苷酶基因LmNAG1的时空表达。运用RT-PCR和Real-time PCR技术对飞蝗β-N-乙酰氨基葡萄糖苷酶基因在不同龄期、五龄5、6、7天六个不同组织部位、四龄到五龄表皮中的表达进行了分析。结果显示该基因在飞蝗各个龄期均有表达,表达量随龄期递增；不同组织部位分析显示该基因在前后肠表达量最高；四龄和五龄表皮中的表达变化与20-E滴度吻合,在蜕皮前两天表达量最高。
三、飞蝗β-N-乙酰氨基葡萄糖苷酶基因LmNAG1的生物学功能研究。对LmNAG1进行RNA干扰实验,结果表明：二龄和五龄飞蝗若虫注射dsLmNAG1后,目的基因被沉默,在各组织部位的表达量均显著降低,沉默持续时间>72h；注射后的若虫表现为蜕皮时间延迟、新旧表皮无法完全分离而死亡
四、飞蝗β-N-乙酰氨基葡萄糖苷酶总酶活的测定分析。对五龄5、6和7天飞蝗不同组织部位及整虫酶活性进行测定;RNA干扰后不同时间点测定β-N-乙酰氨基葡萄糖苷酶总酶活性。结果显示：九龄最后3天该酶活性与其生理过程相关；已获得的LmNAG1基因被沉默后,β-N-乙酰氨基葡萄糖苷酶总酶活性显著下降。
综上所述,本文获得的飞蝗β-N-乙酰氨基葡萄糖苷酶基因在飞蝗生长发育过程中具有重要作用,其生物学功能与飞蝗蜕皮这一生理过程密切相关,该基因的沉默可导致飞蝗因无法完成蜕皮而死亡,且对β-N-乙酰氨基葡萄糖苷酶总酶活性具有显著的影响。该基因对于RNAi介导的飞蝗有效防治具有潜在的应用价值。
β-N-acetylglucosaminidases are widespread in bacteria, fungi and arthropods. There are often multiple genes that encoding P-N-acetylglucosaminidases in various insect species, which involve in different physiological processes including chitin degradation, glycoconjugates degradation and N-glycan modification. Because chitin is an indispensable structural component for insects but absent in higher plants and animals, the β-N-acetylglucosaminidases which is involved in chitin degradation could be an ideal potential target for pest control. The main contents of this thesis are as follows:
1. Cloning, sequencing and phylogenetic analysis of β-N-acetylglucosaminidase in Locusta migratoria.17β-N-acetylglucosaminidase gene fragments from L. migratoria were identified by searching the sequences in the EST database. Fifth-instar nymphs were used for template to obtain full-length sequence of putative LmNAGl. The full-length cDNA consists of2667nucleotides, including an open reading frame (ORF) of1845nucleotides that encode614amino acid residues and233-and589-bp non-coding regions at the5'-and3'-end of the cDNA, respectively. Phylogenetic analysis of the NAGs from L. migratoria and several other insect species showed that LmNAGl was grouped with the enzymatically characterized NAGs in group I.
2. Developmental stage and tissue-specific expression patterns of LmNAGl. RT-PCR and Real-time PCR were carried out to analyze the expression patterns of LmNAGl. Six selected tissues were dissected from day5,6and7of fifth-instar nymphs for tissue-specific expression analysis. The whole insects from the third day of each stage and integument dissected from each day during4th and5th-instar nymph stages of L. migratoria were used for developmental expression analysis. The results showed that LmNAGl was expressed in all the developmental stages and increased along with insect growth. The qPCR analyses showed that LmNAGl was predominantly expressed in foregut and hindgut, relatively low in the integument, midgut and wing pad, but hardly detectable in gastric caeca. The expression of LmNAGl in integument could be detected in whole4th-and5th-instar nymphs. The expression level of LmNAGl was significant high in the last two days of each instar, which is accordant with the ecdysone hormone (20-E) titer peak in both4th-and5th-instar nymphs.
3. Biofunctional analysis of LmNAGl gene. RNAi experiment was carried out to determine the biological function of LmNAGl. The results showed target gene could be down-regulated effectively in all selected tissues after dsLmNAGl injection, and the silence time could be kept more than72hours. The abnormal nymphs showed an "incomplete" ecdysis phenotype after the dsRNA injection and most of them eventually died from failed molting.
4. β-N-acetylglucosaminidase total activity assay. The experiment determined the β-N-acetylglucosaminidase total activity of six selected tissues from day5,6and7of L. migratoria5th-instar nymphs. The results showed that the distribution of the enzyme activity obviously change in the six tissues of the last three days of5th-instar nymphs and the patterns correspond with the process of molting and other crucial physiological functions of insect. The total activity of LmNAG starts to significantly decrease between24h and48h after LmNAGl dsRNA injection.
To summarize, the obtained LmNAGl gene plays a significant role in the developmental process of L. migratoria. Its biological function is closely related with the important process of molting. The knock-down of the gene could cause the mortality of the insect because of incomplete ecdysis and also supress suppress the total enzyme activity of β-N-acetylglucosaminidase in L. migratoria. The results suggest LmNAGl is a potential and promising target for RNAi-mediated biological pest control.
引文
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