MicroRNAs (miRNAs)作为转录后基因表达的一个重要的调控因子，参与了包括发育、代谢、疾病的发生等各种重要的生理过程。家蚕基因组的测序完成为在全基因组水平鉴定家蚕miRNAs以及与其他昆虫进行比较基因组学研究提供了契机。
在中国科学院北京基因组研究所于军研究员的指导下，俞晓敏、周青、蔡亦梅所在小组建立了一套基于Srnaloop及已知动物pre—miRNAs结构特征的miRNA的预测体系；同时，利用miRNAs克隆鉴定平台，对家蚕14个发育时期的miRNAs进行克隆鉴定。利用计算预测结合克隆测序的方法，一共鉴定了118个保守的家蚕miRNAs和151个新的miRNAs。对118个保守的miRNAs分析后发现，有6对进化上保守的miRNAs簇；同时发现，Dicer的不精确和选择性剪切可能赋予miRNAs新的功能。对克隆到的miRNAs在14个家蚕发育时期分布进行分析，发现家蚕四龄蜕皮期miRNA的表达种类和数量上均多于其他发育时期，进一步利用Stem—loop RT PCR分析表明，部分保守的miRNAs在该时期表达量变化比较剧烈，提示miRNAs在家蚕蜕皮期可能起着至关重要的调控作用。在对15个保守的家蚕miRNAs进行表达量分析后，发现其中的8个miRNAs表达量在不同的发育时期变化明显，结合靶基因预测的结果以及家蚕发育阶段的特点，筛选并分析了部分miRNAs的靶基因及其在家蚕发育过程中可能的作用机制，为系统的发掘和阐明miRNAs在家蚕发育过程中的调控机制研究提供理论依据。
目前，该小组正在结合家蚕新公布的精细基因组数据，对家蚕miRNAs进行深度挖掘；同时，基于前期的研究结果，开展miRNAs在家蚕蜕皮期调控机制的研究。该课题将作为我所正在进行的转录组学研究的一个重要组成部分，并为转录组学后续的功能学研究建立良好的技术平台。
相关论文在近期《公共科学图书馆·综合》（PLoS ONE）杂志发表 (2008—8—20)。Yu X，Zhou Q，Li S—C，Luo Q，Cai Y，et al. (2008) The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages. PLoS ONE 3(8): e2997.（生物谷Bioon.com）
生物谷推荐原始出处：
PLoS ONE，3(8): e2997. doi:10.1371/journal.pone.0002997，Xiaomin Yu, Jun Yu
The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages
Xiaomin Yu1,2#, Qing Zhou1,2#, Sung-Chou Li4,5#, Qibin Luo1,3, Yimei Cai1,2, Wen-chang Lin4,6, Huan Chen1,3, Yue Yang1,2, Songnian Hu1, Jun Yu1*
1 Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China,
2 Graduate University of Chinese Academy of Sciences, Beijing, China,
3 James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, China,
4 Institute of BioMedical Informatics, National Yang-Ming University, Taipei, Taiwan,
5 Bioinformatics Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan,
6 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Background
MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species.
Methodology/Principal Findings
We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis.
Conclusions/Significance
Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms.
