【作者】 吴锦雅；

【导师】 陈晓光；

【作者基本信息】 南方医科大学 ， 病原生物学， 2011， 博士

【摘要】 MicroRNA (miRNA)是新近发现并引起广泛关注的一类非编码小RNA分子,它在生物体内发挥重要的基因表达调控作用,是一套全新的表达调控机制。它广泛存在于动物、植物、细菌、病毒等多种生物物种中,在最新的miRBase Virsion16.0中共收录miRNA 15172,成熟1miRNA序列超过17000条,涉及142个物种,其中大多数miRNA在生物进化过程中高度保守。目前发现,1niRNA是生物体内最丰富最重要的一类基因调控因子,在内源基因的调控以及基因组对抗外源核酸侵入中起着重要的作用。在真核生物中,miRNA基因约占整个基因组基因总数的2%,其可能调控的mRNA占全部mRNA的比例高达30%。研究发现miRNAs在细胞凋亡、癌症发生、胚胎发育、干细胞分化、炎症、生长发育、病毒感染等过程中发挥重要作用。研究表明miRNA通过与靶基因mRNA特异性的碱基配对,引起靶mRNA的降解或者抑制其翻译,从而对基因表达进行转录后调控。每个miRNA可以有多个靶基因,而几个miRNAs也可以调节同一个基因。这种复杂的调节网络既可以通过一个miRNA来调控多个基因的表达,也可以通过几个1miRNAs的组合来精细调控某个基因的表达。在线虫、果蝇、小鼠和人等物种已经发现的miRNAs功能研究中,多数具有和其他参与调控基因表达的分子一样的特征,即在不同组织、不同发育阶段中miRNA的水平有显著差异,这种miRNA表达模式具有的位相性和时序性,提示miRNA有可能参与基因表达的调控。正是由于miRNA具有的基因表达调控功能和特点,使其对基因功能研究、生物进化探索和人类疾病防治具有重大意义,因而成为继siRNA (small interfering RNA)之后的新的研究热点。在蚊虫的miRNA研究领域,2005年,Wang等首次运用生物信息学的技术方法,鉴定了38个冈比亚按蚊的miRNAs。2007年,Winter等首次实验验证冈比亚按蚊miRNAs,比较了冈比亚按蚊不同时期的miRNA表达谱,并通过对敲除Dicer1和Agol mRNA(这两种酶与miRNA的生成有关)前后冈比亚按蚊感染疟原虫的生长发育情况表明miRNA作为一类新型的调节因子可能参与按蚊对疟原虫的防御反应。Mead等和Li等人通过实验分别鉴定了斯氏按蚊埃及伊蚊miRNAs,并揭示miRNA可能在生长发育及传播疾病过程中起到重要调节作用。2010年Scalsky等分析致卷库蚊成蚊和白纹伊蚊C7/10细胞感染西尼罗病毒前后miRNA表达差异,提示miRNA可能与病毒感染有关。白纹伊蚊作为登革病毒传播重要的传播媒介之一,主要分布在我国南部和东南亚地区。近年来登革热病例正在迅猛增加,其流行已威胁到全球二分之一人口的健康安全。在我国,登革热的流行以广东省最为严峻。自1978年广东佛山首次暴发登革热以来,广东省已发生过12次大小不等的登革热流行,近年来更常有暴发流行。登革热在广东的流行,不仅给广大人民群众的生命健康带来了严重危害,而且还经常转化为突发的公共卫生事件,给社会带来不安定,并严重影响经济的发展。由于目前还没有有效的疫苗可用于登革热的预防,预防登革热只能从控制传染源和传播媒介方面着手,而控制传染源难度较大,故此,而今预防登革热的唯一对策就是控制其传播媒介----埃及伊蚊和白纹伊蚊,但目前这方面的基础研究,如蚊虫对登革病毒的易感性和传播以及现代生物技术在蚊媒及其传染病防治中的应用,还很欠缺、亟待加强。因此,弄清楚白纹伊蚊对登革病毒的易感性基础对于制定有针对性的防制措施非常重要。白纹伊蚊miRNA是否存在?其序列构成如何?有何表达特征?在登革病毒的感染过程中起什么作用?能否从中找到阻断登革热传播的靶标呢?在本研究开始之前,有关白纹伊蚊miRNAs研究尚未见报道。本研究利用新一代Solexa高通量测序技术,通过对白纹伊蚊miRNAs的组学特征以及miRNAs在白纹伊蚊不同发育阶段的表达特征进行深入分析。Solexa测序是Illumina公司推出的新一代高通量、大规模测序技术,具有高准确性、高通量、高灵敏度、和低运行成本等突出优势,目前已广泛应用于基因组测序、转录组测序以及小RNA测序领域。其中在miRNA研究领域,通过小分子RNA大规模测序分析,可以从中获得物种全基因组水平的miRNA图谱,实现包括新miRNA分子的挖掘,其作用靶基因的预测和鉴定、样品间差异表达分析、miRNAs聚类、进化分析以及表达谱分析等科学应用。因为这一测序技术不需要依赖于基因组序列,因此可以对目前尚无基因组序列的物种进行测序分析,拓宽了miRNA的研究领域,既极大的丰富了miRNA的种类,又为miRNA的结构特点以及功能的研究提供了便利。在本课题开展过程中,人们通过Solexa高通量测序技术对鸡胚、家蚕、蜜蜂、蝗虫、库蚊和人等多个物种的小RNA进行了深度测序,极大的丰富了miRNA的种类,促进了miRNA的深入研究。本课题利用新一代高通量测序和生物信息学的技术方法,首次对白纹伊蚊miRNA的组学进行系统性分析,并将其组学特征与果蝇、按蚊的miRNA进行比较分析；应用Solexa和Northern blot分析miRNA在白纹伊蚊不同生长发育阶段的表达特征,为进一步探索miRNA在蚊虫生长发育过程中的调控机制提供依据；应用Solexa测序技术与生物信息学方法分析经登革病毒感染前后的miRNA表达差异及其靶基因,为登革病毒调控机制的研究提供新的思路和方法。研究目的：1.通过新一代高通量测序技术Solexa测序和生物信息学的技术方法,对白纹伊蚊miRNAs的组学进行系统性分析,丰富蚊虫miRNA并揭示新的蚊虫特异miRNA。2.应用Solexa测序和Northern blot对miRNA在白纹伊蚊不同生长发育阶段的表达特征及种系进化分析,为进一步探索miRNA在蚊虫生长发育过程中的调控机制提供依据。3.应用Solexa测序技术与生物信息学的方法分析经登革病毒感染前后的miRNA表达差异及预测其靶基因,初探调控登革病毒靶标,为研究登革病毒感染调控机制奠定基础研究方法：1高通量测序及其信息学分析白纹伊蚊miRNA方法的建立：1.1样品收集：分别收集12-24h虫卵、早期幼虫、晚期幼虫、虫蛹、羽化3-5d雄蚊、雌蚊等6个不同阶段样本。1.2 Total RNA提取和鉴定：用Trizol提取各个不同阶段样本的Total RNA,核酸测量仪检测浓度及纯度,15%尿素变性聚丙烯酰胺凝胶电泳鉴定所提Total RNA的质量,将6个样品total RNA等量混匀后用Agilent生物分析仪分析RNA完整性。1.3传统方法建库及验证：采用TA克隆和传统Sanger测序对所提Total RNA鉴定是否可以用于Solexa测序。1.4 Solexa高通量测序：首先将18-30nt范围的small RNA从Total RNA中分离出来,两端分别加上特定接头后体外反转录成cDNA,经RT-PCR扩增后用Solexa测序仪对DNA片段进行单向末端直接测序。1.5原始序列的处理与统计：通过去接头,去低质量,去污染、统计序列长度分布等过程,获得去杂质的“干净”序列(Clean序列)。1.6小RNA序列的比对分析：将上述clean序列通过与NCBI Genbank (http:www.ncbi.nlm. nih.gov/)中rRNA、tRNA、snRNA、snoRNA、repeat、exon、intron、miRNA等库进行比对和分类注释。1.7已知1miRNA鉴定和新miRNA预测：用SOAP软件选择没有比对任何注释的小RNA与埃及伊蚊基因组序列比对,Ensembl法(http://www.ensembl.org)获得了与埃及伊蚊吻合序列及其100nt侧翼基因序列,用MIREAP软件鉴定和预测miRNA,最后用RNAfold(http://ma.tbi.univie.ac.at/cgi-bin/ RNAfold.cgi)分析其最低能量构象。1.8用Northern blot方法验证Solexa测序技术分析鉴定miRNAs2白纹伊蚊IniRNAs组学的鉴定：2.1用上述建立的Solexa高通量测序和生物信息学分析虫卵、幼虫、蛹、雌雄成虫及吸血后的6个发育阶段miRNAs。2.2将鉴定miRNAs与miRBase中库蚊、按蚊、果蝇、意蜂、家蚕、原鸡、斑马鱼、小鼠和人等物种miRNA进行种系进化分析,并将获得的miRNAs与三个蚊种基因组序列进行比对。2.3 miRNA基因簇分析：以Altuvia等的miRNA成簇分析方法为基础加以改善进行miRNA成簇分析。以1000nt作为相邻miRNA间距离的最大阈值来寻找染色体上同一方向可成簇miRNA。3白纹伊蚊miRNAs表达谱分析及Northern blot验证：3.1 miRNA差异表达分析：根据Solexa测序结果,统计白纹伊蚊miRNA在各个阶段表达量,并归一化。然后以未吸血雌蚊为参照,制1og2-rato、Scatter plot图,比较2个样品中miRNA表达差异。3.2 miRNA表达谱分析：将6个发育阶段各个miRNA求平均值,用MeV软件聚类分析各个发育阶段miRNA表达。3.3 Northtern blot验证对不同发育阶段样本及吸血前后白纹伊蚊miRNA的表达谱分析。4登革病毒感染后白纹伊蚊miRNA的Solexa分析：4.1 Solexa测序方法对白纹伊蚊感染登革病毒前后表达差异分析,寻找特异性表达miRNA。4.2 RNAhybrid软件预测针对登革病毒基因组的miRNA。研究结果：1通过对白纹伊蚊5个发育阶段混合样品的Solexa测序以及生物信息学分析,共测得序列12663936条。按照rRNAetc->repeat -> exon -> intron>known miRNA-的优先级顺序将小RNA遍历,分别是5094481、986、51020、307750、2368768。进一步鉴定79种已知miRNAs,6种新的miRNAs。2 Northern blot验证miR-184在各个期均有表达,而miR-2941仅在虫卵中表达,证明Solexa高通量测序方法的可靠性。3用上述建立的Solexa高通量测序方法对白纹伊蚊虫卵、幼虫、蛹、雌雄成虫及吸血后的雌蚊6个发育阶段miRNAs组学分析,共获得已知91种miRNAs,37个新miRNAs。4将获得91种已知miRNAs进行种系进化分析,发现白纹伊蚊与昆虫类miRNA在进化上比脊椎动物更为保守,其中与库蚊miRNA同源性最高。5 37个新miRNA是蚊虫特有的,进一步与冈比亚蚊、致卷库蚊基因组序列比对,其中miR-B-m0097, miR-F-m0156和miR-M-m0019在按蚊基因组序列中发现,在库蚊中除了上述miRNA外,miR-2941、miR-2943、miR-M-m0036等10种miRNA在库蚊发现,其余26个均为伊蚊特异miRNA.6 128个miRNA中19个pre-miRNAs是高度相似或相同的,如miR-137-1和miR-137-2、miR-263a和miR-263b、miR-2941-1和miR-2941-2等。7共发现10个白纹伊蚊基因簇,其中8个基因簇各个簇的miRNA距离小于lkb。基因簇aal-miR-306b、aal-miR-79、aal-miR-9b共同位于染色体CH477970.1,已经明确编码基因功能即丝氨酸苏氨酸激酶的内含子上miRNA簇。8 128miRNA在白纹伊蚊各个生长发育阶段聚类分析显示未成虫和成虫2个大类,其中未成虫中包括虫卵、幼虫和蛹,其中蛹和幼虫阶段的miRNA表达特征上更为接近。从纵向miRNA在各个阶段表达分析,共有虫卵特异性、幼虫特异性、蛹特异性、雄虫特异性、雌虫特异性及吸血后雌虫+幼虫特异性6个簇。其中虫卵和雄虫显示明显期特异性表达。31miRNAs在虫卵中呈高表达,如miR-315、miR-2941、miR-2943、miR-2944、miR-2945、miR-E-m0165等。54miRNAs在雄虫中呈高表达,其中某些miRNA在雌虫中也呈高表达如let-7、miR-210等。9 Northern blot对6种保守性1niRNAs与6种蚊虫特异性miRNAs进行验证。所有的白纹伊蚊miRNAs至少在一个发育阶段出现20nt左右的条带,并且除了个别miRNAs表达略有不同外,其与miRNAs表达谱与Solexa测序结果高度相似。10 Solexa高通量分析吸血前后的miRNAs表达差异分析,大部分miRNAs在吸血前后表达量保持一致,miR-263、miR-283、miR-308、miR、miR-92、miR-998、miR-989、miR-2941、miR-2944、miR-184吸血后表达量增加,miR-B-m003、miR-7、miR-993、miR-957在吸血后表达量降低。进一步northern blot分析4种miRNAs (miR-184、miR-998、miR-989、miR-2941)均在吸血后表达水平增加,与Solexa结果高度吻合。11经Solexa测序,感染和对照样本中均可检测到91种保守1niRNAs和37种新miRNAs。白纹伊蚊经登革病毒感染后miRNA表达谱差异显著,其中38个白纹伊蚊miRNAs经登革病毒感染后的表达下调,21个miRNAs经登革病毒感染后表达上调。12用RNAhybrid软件和SMVLight软件筛选登革病毒靶基因,结合表达谱差异,共获得23种登革病毒感染后差异表达的miRNAs,其靶基因分别针对登革病毒基因组非结构蛋白(1、2、4、5)和包膜蛋白基因等。研究结论：1首次用Solexa高通量测序技术并结合生物信息学方法对白纹伊蚊虫卵、幼虫、蛹、雌雄成虫及吸血后的雌蚊6个生长发育阶段miRNAs组学进行系统性分析,丰富蚊虫miRNA,并揭示新的蚊虫特异的miRNAs。2白纹伊蚊系统性表达谱分析表明白纹伊蚊miRNAs在不同发育阶段表达具有期特异性。二者结果高度相似性,进一步验证了Solexa高通量测序技术可作为miRNA表达谱分析的可靠性。3雌蚊吸血前后的miRNAs表达差异表明miRNA与雌蚊的生长发育和吸血有关,对其功能及作用机制的研究将有助于我们对蚊虫繁殖、发育及疾病传播的进一步了解。4经登革病毒感染后白纹伊蚊miRNA表达谱差异显著,提示miRNA可能再在抗病毒、抗感染中起着重要的调控作用。为研究登革病毒感染调控机制奠定基础。更多还原

【Abstract】 BackgroundMicroRNAs (miRNAs) are～22-nucloetide RNAs that modulate gene expression by targeting cognate mRNAs for cleavage or translational repression. They are endogenous and widely distributed in metazoans and plants. In the latest MirBase vision16.0, there are 15172 miRNAs in 142 species and most miRNAs are highly conserved across divergent species. Estimates of the extent of miRNA gene regulation vary from 4% of transcripts in the Drosophila ovary to a third of human genes. At present miRNA is the most important regulator and they have been shown to be involved in the regulation of many biological processes including apoptosis, cancer, embryonic development, immunity and infection.Mature miRNAs associate with an Argonaute protein and bind their mRNA targets, which are often in the 3’untranslated region (UTR), resulting in inhibition of translation or possibly target mRNA degradation in animals. Each miRNA may have multiple target genes and several miRNAs can regulate the same gene simultaneously. Functional studies of miRNAs in C. elegans, Drosophila, mouse, human and other species suggested that the amount of miRNAs varied significantly in different tissues or at different developmental stages, similar to many other molecules involved in gene expression regulation. The specific temporal and spatial expression of miRNAs suggested that miRNA might be involved in gene regulation. Because the miRNA plays an important role in gene regulation, it becomes a new hotspot after small interfering RNA in studies of gene function, biological evolution and disease control. For mosquitos, in 2005, Wang et al, for the first time, identified 38 miRNAs in Anopheles gambiae by computational prediction of similarity to known miRNAs from other species. In 2007, Winter et al. experimentally identified 18 miRNAs from Anopheles gambiae including three miRNAs unique to mosquito. They used a combination of shot-gun cloning and bioinformatic analysis method. In addition, they found that Dicer1 and Agol mRNAs knocked out A. gambiae was more sensitive to the infection of Plasmodium, suggesting that miRNAs might be involved in the defence reaction of Anopheles. Mead and Li also experimentally identified miRNAs in An. stephensi and Ae. Aegypti, as well as finding that miRNA played a regulatory role in development and disease transmission. In 2010. Scalsky infected Culex mosquitoes with WNV and two miRNAs, miR-92 and miR-989, and resulted in significant changes of miRNA expression levels following WNV infection, suggesting that miRNAs might be linked to virus infection in mosquitos.Aedes albopictus, as the main vector of Dengue virus, is widely distributed in southern China and Southeast Asia. Rapid increase of Dengue fever (DF) incidence in recent years has made it a serious public health threat to nearly half of the world’s population. In recent years, Guangdong province has had the highest number of DF epidemics, with cases reported every year since 1997. The prevalence of dengue fever in Guangdong, not only poses a serious health hazard, but also often translate into sudden public health events, which brought society instability and serious impact on economic development. As no DF vaccine is currently available, the only effective means for DF prevention is mosquito vector including Aedes aegypti and Aedes albopictus, but basic research such as mosquito susceptibility to dengue virus and spread as well as Modern biological technology in mosquito vectors and infectious disease control application, is still lacking. Therefore, it’s very important for control and prevention of dengue fever to discover Aedes albopictus the susceptibility.Before we carryed out the present study, there are no answeres for the following questions:(1) Do miRNAs exist in A.albopictus? (2) What are the sequences of miRNA in A.albopictus and what’s their expression pattern? (3) If miRNA play any role in the infection of dengue virus and is there any possiblity to block dengue transmission by targeting on miRNA?The present study employes the highthroughput sequencing capacity provided by Solexa GA to study the OMIC of miRNA in A.albopictus, and explores detailed expression pattersn of miRNAs at different developmental stages. Solexa Genome Analyzer is one of next -generation sequencing technologies produced by Illumina, it has priorities of high accuracy, high throughput, high sensitivity and cost-effective. The method has been widely used in genome sequence, transcriptome sequence and small RNA sequencing studies. Determining miRNAs using Solexa hasa high potential to identify novel miRNA as well as determining the experssion profiles of known and novel miRNAs. The Solexa sequencing technique does not need to clone a library and the sequencing read length is compatible with the length of mature miRNAs. These characters made the Solexa sequencing particularly useful for studying miRNA. The experimental process is very simple and cost-effective, and the method dramatically increases the throughput and has a deeper coverage. To date, small RNA in chicken somites, Bombyx mori, Apis mellifora, Locust and culex were identified by Solexa sequencing which expanded the list of miRNAs and accelerated of the study of small RNAs. In the present study, we performed the first systhematic analysis of miRNAs in Aedes albopictus using a combination of Solexa high throughput sequencing and bioinformatics analysis, and compared with miRNAs in other species according to miRBase virsion14.0. Our result substantially expanded the list of miRNAs for mosquitoes and uncovered a number of novel and mosquito-specific miRNAs. Northern blot and Solexa sequencing found that some miRNAs changed during mosquito development and blood feeding. Using Solexa high throughput sequencing and bioinformatics approaches, we analyzed the expression difference of miRNA in Ae.albopictus infected with dengue virus and predicted targets of these miRNAs in dengue virus’s genes, providing potential clues for regulation mechanism of dengue virus in mosquitoes.Objective 1. Analyze miRNAs in Aedes albopictus using Solexa high throughput sequencing and bioinformatics approaches, enrich the list of mosquito miRNAs, and find novel and mosquito -specific miRNAs.2. Analyze expression patterns of miRNAs in different developmental stages in mosquito using Solexa sequencing and northern blot technologies, and find miRNAs that play potential roles during development and blood feeding activies.3. Analyze expression difference of miRNA in Ae.albopictus between infected and uninfected by dengue virus and predicted target genes of miRNAs in dengue virus using Solexa high throughput sequencing and bioinformatics approaches.Methods1 Developing methods for determining miRNAs in Aedes albopictus by Solexa sequencing and bioinformatics analysis:1.1 Sample collection:Six samples were prepared for solexa sequencing, including embryo, larvae, pupa, male mosquito, female mosquito.1.2 Extraction of total RNA and quality control:All samples were firstly frozen in liquid nitrogen and total RNA were isolated according to the user instruction of Trizol. The concentration of RNA was determined using nucleic acid quantitator. The purity was analyzed by 15% denaturing polyacrylamide gel and Agilent Bioanalyzer.1.3 Identification of small RNA by the traditional clone library and sequencing method:Using TA clone to construct library and sequenced by the traditional Sanger capillary sequencing method1.4 Solexa sequencing:After detecting purity with Agilent Bioanalyzer, the 18-30nt small RNAs were isolated from total RNA, the 3’and 5’termini of RNA molecules are joined with special adapters respectively and are reversely transcribed into cDNA library for single-end sequencing.1.5 Raw data analysis:The low quality tags, adaptors, and contaminations formed by adaptor-adaptor ligation were removed and the residual reads were analyzed. 1.6 SmallRNAs maping:All sequences were annotated through the alignment with rRNA, tRNA, snRNA, snoRNA, repeat and degradation fragments of exon or intron. The residual sequences were labled as unannotated and were used to predict the novel miRNA.1.7 Identification of known miRNA and prediction of novel miRNA:Clean reads were aligned against the Ae. Aegypti genome (Ae. Albopictus genome has not sequenced so far and Ae. Aegypti is the most closely related species) using SOAP. Sequences with perfect match or one mismatch were retained for further analysis. To further analyze the RNA secondary structures,100 nucleotides of genomic sequence flanking each side of matched sequences (potential miRNAs) were extracted, and the secondary structures were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) and analyzed by MIREAP (http://sourceforge.net/projicts/mireap)using default settings.1.8 Identification of miRNAs by northern blot.2 Systhematic analysis of miRNAs in Aedes albopictus:2.1 Analysis of miRNAs in egg, lava, pupa, adult, and blood female in Aedes albopictus by Solexa sequencing and bioinformatics analysis2.2 Comparison with Culex, Drosophila, Apis mellifera, Bombyx mori, Gallus gallus, Danio rerio, Mus musculus, Humus sapient in miRBase.2.3 Clustering of miRNA genes:Find miRNA cluster based on the cutoff in 1000nt in one chromosomeaccording according to method of Altuvia.3 Expression profiles of miRNAs in Aedes albopictus analyzed by Solexa sequencing and northern blot:3.1 Expression difference of miRNAs:The total RNAs from embryo, larvae, pupae, male adult, female adult fed sugar and blood were used for solexa sequencing respectively, and the protocol described as above. One hundred twenty one miRNAs from each sample were analyzed by Solexa reads. All expression data are normalized as following:Expression of normalization=Expression of miRNA /expression of total miNRA from sample xnormalization coefficient. With reference to female, compare miRNAs expression with other samples. 3.2 Expression profiles at different developmental stages were analyzed with MeV by hierarchical clustering.3.3 Identification of expression profiles in different stages in mosquito by northern blot.4 Analysis of miRNAs in Ae. Albopictus infected by dengue virus by Solexa:4.1 Comparison of miRNAs infected and uninfected mosquito and exploration of dengue-specific miRNAs.4.2 Prediction of target genes of miRNAs on dengue virus genome by RNAhybrid.Results1 After filtering linker sequences and ambiguous reads, a total of 12663936 clean small RNA reads were obtained for the mixed sample of multiple developmental stages. We aligned sequencing reads to rRNAs, repeat, exon, intron and known miRNA used by NCBI GenBank dabase (http:www.ncbi.nlm.nih.gov/), and there are 5094481、986、51020、307750.2368768 reads espectively.2 The 79 known miRNAs and 6 novel miRNAs were identified by SOAP and MIREAP and all of them have hairpins with length of 70bp.3 Northern blot determined miR-184 in each developmental stage, and miR-2941 only in egg stage, supporting the Solexa high-throughputing result.4 91 known miRNAs and 37 novel miRNAs in 6 developmental stages were uncovered.5 Comparison of known miRNAs in Ae.albopictus with other species showed that, although the known miRNAs are highly conserved throughout widely divergent animal taxa, the pattern of Ae.albopictus miRNA is more similar to insect than vertebrates.6 Comparison of novel miRNAs in Culex and Anopheles gambiae genomes showed that miR-B-m0097, miR-F-m0156 andmiR-M-m0019 was found in Anopheles gambiae, and 10 novel miRNAs such as miR-2941, miR-2943, and miR-M-m0036 were in Culex, while the rest are Aedes-specific.7 There are 19 out of 128 total miRNAs that were highly similar, such as miR-137-1 and miR-137-2, miR-263a and miR-263b, miR-2941-1 and miR-2941-2, etc.8 There were 8 clusters of miRNAs that showing more than one miRNA hairpin sequences within 1 kb. A miRNA gene cluster within an intron of a gene coding for a serinethreonine kinase group protein. The miRNA gene cluster contained miR-306b, miR-79 and miR-9b.9 The heatmap showed a systematic overview of expression patterns of 129 miRNAs. Clustering of the 6 samples showed that the adult samples (male and sugar-fed and blood-fed females) formed a group, while the other 3 developmental stages formed the other one. We observed obvious stage specific expression of miRNAs in different developmental stages. Hierarchical clustering showed many miRNAs were highly expressed in either the embryo (cluster 1) or male (cluster 4) stages.31 miRNAs were highly expressed in the embryo, among them the mosquito specific miRNAs including miR-2941, miR-2943, miR-2944, miR-2945, and miR-E-m0165. There were 54 miRNAs highly expressed in the male adult, besides some of them were also expressed in female adults. The northern blot of let-7, miR-210, miR-1890, miR-1891 also proved that those miRNA were mainly expressed in the lateral developmental stages, but not being expressed before the pupae stage.10 We further determined 6 conserved miRNAs and 6 mosquito specific miRNAs using northern blot to confirm the illumina sequencing results. All 12 miRNAs showed positive expression, at least at one of the 5 developmental stages, with signals at～22 nt by northern blot. Grossly, the presence/ absence patterns in 5 developmental stages are consistent to the illumina results for all determined miRNAs.11 The expression of a large proportion of miRNAs (70) was increased after blood-feeding, and some (29) were dysregulated. The increasing expression of miR-2941, miR-184, miR-998, and miR-989 has also being proved by northern blot data.12 The expression of a large proportion of miRNAs has changed after infected by dengue virus. There are 38 miRNAs decreased and 21 miRNAs increased after the infection of dengue virus.13 According to the target prediction and expression profile,23 target genes were identified on dengue genes, aiming at non-structural proteins (1,2,4,5) and envelope proteins.Conclusions1 Using Solexa high throughput sequencing and bioinformatics approaches, we performed the first systhematic analysis of miRNAs in Ae. Albopictus, substantially expanded the list of miRNAs for all mosquitoes, and uncovered a number of novel mosquito-specific miRNAs.2 Expression profiles of miRNAs in Ae. Albopictus suggested that several miRNAs may play important roles in the development. High similar result between Solexa and northern bolt demonstrated that Solexa sequencing have a high accuracy.3 Expression levels of several miRNAs have changed after blood feeding, indicatingthat they might be related with embryonic development and reproduction. The result could be helpful for understanding disease transmission in mosquitos.4 Expression of miRNAs was altered after infectionof dengue virus, suggesting that miRNAs may play important regulation roles inviral infection in the mosquito host.更多还原