飞蝗Argonaute1的分子特性及生物学功能

doi:10.3864/j.issn.0578-1752.2016.18.011

摘要/Abstract

摘要： 【目的】MicroRNAs(miRNAs)是一类长度约22 nt的非编码RNA，通过转录后调控的方式在多种生命活动中发挥重要功能。Argonaute1（AGO1）蛋白作为miRNA沉默复合物（miRNA silencing complex RISC）的重要组成部分，在miRNA调控通路中起着关键作用。论文旨在研究AGO1的生物学功能及其对飞蝗（Locusta moratoria）生长发育的影响，为探索昆虫miRNA的生物合成和农业害虫的有效控制提供理论依据。【方法】采用生物信息学方法在飞蝗转录组数据库中获得LmAGO1 cDNA序列；使用在线蛋白翻译软件（ExPASy）对LmAGO1进行蛋白翻译，利用SMART分析LmAGO1蛋白的功能结构域；选取家蚕（Bombyx mori）、果蝇（Drosophila melanogaster）和赤拟谷盗（Tribolium castaneum）等模式昆虫的同源序列与LmAGO1氨基酸序列进行聚类分析，采用Phyml软件构建昆虫AGO蛋白的系统发育树；为了进一步研究LmAGO1在飞蝗生长发育过程中的作用，使用T7 RiboMAXTM Express RNAi System体外合成LmAGO1的dsRNA，在飞蝗4龄第2天和5龄第2天若虫期连续两次注射dsRNA进行干扰，同时注射dsGFP作为对照。分别收集注射dsRNA后48 h和72 h的整虫样品提取RNA，反转录为cDNA。通过实时荧光定量PCR（RT-qPCR）检测LmAGO1在不同时间点的干扰效率并观察虫体的发育表型。同时，为了检测LmAGO1沉默是否会影响miRNA的生物合成，采用RT-qPCR对飞蝗体内5个高丰度miRNA表达进行定量分析。【结果】LmAGO1蛋白含845个氨基酸，具有典型的AGO蛋白家族保守结构域，即位于213—348位点的PAZ结构域和502—804位点的PIWI结构域。聚类分析表明，LmAGO1蛋白与其他昆虫的AGO1蛋白聚为一类。通过AGO1氨基酸序列同源比对结果显示LmAGO1与模式昆虫果蝇、家蚕AGO1的氨基酸序列一致度高达82.2%和86.9%。RNAi结果表明，虫体注射dsLmAGO1 48 h和72 h后，与对照组相比，LmAGO1表达量均显著降低，干扰效率分别为88.1%和93.0%；进一步观察试虫生长发育的表型特征，与对照组相比，飞蝗4龄期注射dsLmAGO148 h后其生长发育并没有出现明显异常，待蜕皮发育至下一龄期（即5龄期）时，出现大量死亡，死亡率为89.3%；荧光定量PCR结果显示注射dsLmAGO1后，飞蝗体内miRNA-252和miRNA-8的表达显著下降，干扰72 h后miRNA-7、let 7、miRNA-252、miRNA-8的表达均显著下降。【结论】飞蝗AGO1除参与RSIC的形成以外，还可能参与miRNA的剪切加工过程进而调控飞蝗的正常发育。

关键词: 飞蝗, miRNA, Argonaute1, RNA干扰, 生物学功能

Abstract: 【Objective】 MicroRNAs (miRNAs) are small (approximately 22 nt), noncoding RNA molecules that play important roles through post-transcriptional regulation in a wide range of biological process. As an essential component of miRNA silencing complex (RISC), Argonaute1 (AGO1) protein plays a key role in miRNA regulatory pathway. The purpose of this study is to explore the biological function of LmAGO1, and determine its impacts on growth and development in the migratory locust. These results will provide an important theoretical basis for biogenesis mechanism of insect miRNAs and effective pest control. 【Method】 The cDNA sequence of LmAGO1 was identified from the locust transcriptome database by using bioinformatics approaches, and was translated into protein sequence using online protein translation software (ExPASy). The conserved domains were analyzed by SMART based on the deduced protein sequence. A phylogenetic tree of insect AGO was constructed with the locust AGO1, the homologous amino acid sequences from Bombyx mori, Drosophila melanogaster, Tribolium castaneum and other insects using the Phyml program. The double-strand RNAs (dsRNAs) of AGO1 gene were synthesized in vitro using T7 RiboMAX^TM Express RNAi System. The RNA interference (RNAi) experiment was preformed to explore the biological function of LmAGO1 during growth and development in locusts. The dsLmAGO1 was injected into the locust nymphs on day 2 of the 4th-instar and 5th-instar stages, respectively. The injection of dsGFP was used as the control. At the 48 h and 72 h after dsRNA injection, the whole body of locust nymphs was collected for total RNA isolation and cDNA synthesis. The reverse transcript quantitative PCR (RT-qPCR) analyses of the LmAGO1 were performed to determine the gene silencing efficiency. In order to evaluate the influences of the LmAGO1 RNAi on miRNA biogenesis, the five abundantly expressed miRNAs were selected to quantify their expression level by using RT-qPCR after dsLmAGO1 injection.【Result】 The amino acid analysis showed that LmAGO1 protein of locust contains 845 amino acids and has the typical AGO protein family conserved domains, including PAZ (Piwi-Argonaute-Zwille) domain which is located in the region from nucleotide 213 to 348 and PIWI (P-element Induced Wimpy Testis) domain which is located in the region from nucleotide 502 to 804. The phylogenetic analysis showed that LmAGO1 was clustered with the AGO1s of other insect species. The multiple protein sequence alignments indicated that the amino acid sequence identity is 82.2% between LmAGO1and DmAGO1, and is 86.9% between LmAGO1 and BmAGO1, respectively; RNAi results showed that the expression of LmAGO1 was significantly reduced at 48 h and 72 h after dsLmAGO1 injection into locust nymphs. The silencing efficiency of LmAGO1 was 88.1% and 93.0%, respectively. The locust nymphs with dsLmAGO1 treatment did not show any obvious phenotypic defects at the 4th-instar stage. However, compared with dsGFP injected control, the dsLmAGO1 treatment resulted in a high mortality at the 5th-instar stage. A total of 89.3% locust nymphs were dead in dsLmAGO1 injected group. The RT-qPCR results of LmAGO1 showed that LmAGO1 was significantly silenced at 48 h and 72 h after dsRNA injection. Moreover, the expression analysis of miRNA results indicated that the expression levels of miRNA-252 and miRNA-8 were significantly decreased at 48 h after dsLmAGO1 treatments, and that the expressions of miRNA-7, let-7, miRNA-252 and miRNA-8 were significantly down-regulated at 72 h after dsLmAGO1 treatments. 【Conclusion】Besides the role in RSIC formation, LmAGO1 can enhance the processing of miRNA maturation and regulate the normal development in the migratory locust.

Key words: Locusta moratoria, miRNA, AGO1, RNAi, biological function

王艳丽，杨美玲，宋天琪，马恩波，张建珍. 飞蝗Argonaute1的分子特性及生物学功能[J]. 中国农业科学, 2016, 49(18): 3569-3577.

WANG Yan-li, YANG Mei-ling, SONG Tian-qi, MA En-bo, ZHANG Jian-zhen. Molecular Characterization and Biological Function of Argonaute1 in Locusta migratoria[J]. Scientia Agricultura Sinica, 2016, 49(18): 3569-3577.

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