MicroRNAs (miRNAs)作為轉錄后基因表達的一個重要的調控因子,，參與了包括發(fā)育、代謝,、疾病的發(fā)生等各種重要的生理過程,。家蠶基因組的測序完成為在全基因組水平鑒定家蠶miRNAs以及與其他昆蟲進行比較基因組學研究提供了契機。
在中國科學院北京基因組研究所于軍研究員的指導下,，俞曉敏,、周青、蔡亦梅所在小組建立了一套基于Srnaloop及已知動物pre—miRNAs結構特征的miRNA的預測體系,；同時,，利用miRNAs克隆鑒定平臺，對家蠶14個發(fā)育時期的miRNAs進行克隆鑒定,。利用計算預測結合克隆測序的方法,，一共鑒定了118個保守的家蠶miRNAs和151個新的miRNAs。對118個保守的miRNAs分析后發(fā)現,，有6對進化上保守的miRNAs簇,；同時發(fā)現，Dicer的不精確和選擇性剪切可能賦予miRNAs新的功能,。對克隆到的miRNAs在14個家蠶發(fā)育時期分布進行分析,，發(fā)現家蠶四齡蛻皮期miRNA的表達種類和數量上均多于其他發(fā)育時期，進一步利用Stem—loop RT PCR分析表明,，部分保守的miRNAs在該時期表達量變化比較劇烈,，提示miRNAs在家蠶蛻皮期可能起著至關重要的調控作用。在對15個保守的家蠶miRNAs進行表達量分析后,，發(fā)現其中的8個miRNAs表達量在不同的發(fā)育時期變化明顯,，結合靶基因預測的結果以及家蠶發(fā)育階段的特點，篩選并分析了部分miRNAs的靶基因及其在家蠶發(fā)育過程中可能的作用機制,，為系統(tǒng)的發(fā)掘和闡明miRNAs在家蠶發(fā)育過程中的調控機制研究提供理論依據,。
目前，該小組正在結合家蠶新公布的精細基因組數據,，對家蠶miRNAs進行深度挖掘,；同時,，基于前期的研究結果，開展miRNAs在家蠶蛻皮期調控機制的研究,。該課題將作為我所正在進行的轉錄組學研究的一個重要組成部分,，并為轉錄組學后續(xù)的功能學研究建立良好的技術平臺。
相關論文在近期《公共科學圖書館·綜合》（PLoS ONE）雜志發(fā)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）
生物谷推薦原始出處：
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.
