团头鲂let-7 microRNA对其生长和性腺发育的共同调控作用及其分子机制

As a main aquaculture species in the Chinese freshwater polyculture system with high economic value, the blunt snout bream (Megalobrama amblycephala) is gradually exhibiting early sexual maturity under a long cultured period. The growth and gonadal development of early mature individuals exhibit mutual promotion and inhibition relationships, with fast growth and gonad development during the first year, however the growth rate during the second year decreasing significantly. Let-7 microRNA has been demonstrated to play the important functions in the growth and gonad development of mammals. Based on the small RNA and transcriptome data of M. amblycephala from high-throughput sequencing technology in our research team, this project will identify the key let-7 microRNA members which have the main regulation effects on the growth and gonadal development of M. amblycephala. Through the analysis of key let-7 microRNA, its pre-miRNA and targeted genes, the regulation mechnism of key let-7 microRNA will be analyzed. Using artificial synthetic miRNAs and its anti-miR miRNA to over-express and knockdown the key let-7 miRNA activity on cultured cells in vitro, its functions will be deeply analyzed. Morever, the SNP of the key let-7 miRNA and its targeted genes will be identified and the correlations of the SNP with growth and gonadal deveopment of M. amblycephala will be analyzed using selected families in our research team. The results obtained from this study will demonstrate the regulation mechanism of let-7 microRNA on the growth and gonadal development of M. amblycephala and make understanding on their mutual promotion and inhibition relationships. The identified SNP would facilitate M. amblycephala selective breeding program.

团头鲂为我国重要经济鱼类,在长期人工养殖中,性早熟现象明显。性早熟个体生长和性腺发育表现出相互促进-相互抑制关系，1龄阶段生长快、性腺发育快,而2龄阶段生长明显减慢,严重影响其产量。Let-7 miRNA在调控动物生长和性腺发育过程中发挥着重要作用。本研究基于课题组团头鲂小RNA组和转录组高通量测序数据,筛选let-7 miRNA家族中调控生长和性腺发育的关键miRNA,从关键miRNA、其前体及靶基因三个层次系统解析miRNA的作用与调控机理。应用人工合成miRNA及反义miRNA,在细胞水平实现其过表达及沉默, 深入分析关键miRNA的功能。基于课题组团头鲂选育家系,筛选与生长和性腺发育相关联的miRNA 靶标SNP位点。研究结果将解析let-7 miRNA对团头鲂生长和性腺发育的调控机制,深化理解鱼类生长和性腺发育之间相互促进-相互抑制关系,筛选到的关联SNP可应用于其遗传选育研究。

Let-7 miRNA在调控动物生长和性腺发育过程中发挥着重要作用。本项目围绕团头鲂性早熟引起生长速度减慢的问题，探讨let-7 miRNA成员对团头鲂生长和性腺发育的调控作用。（1）在团头鲂上建立其小RNA组测序文库，共筛选到347个已知miRNA以及22个新miRNA。（2）针对let-7 miRNA家族展开系统进化分析，在let-7a/b/c/d/e/f/g/h/i/j10个成员中，let-7a的前体序列与祖先序列最为接近，而其它成员均来自于let-7a的后期分化。（3）定量分析了10个let-7 miRNAs团头鲂雌雄性成熟个体、以及3、6、12月龄生长快慢个体中的相对表达量。其中let-7a在所有的组织中表达量均最高，其次为let-7b和let-7d。在不同生长阶段中，let-7 miRNAs在生长慢组个体的脑和垂体中的表达显著高于生长快组。而let-7a/b/c/d/e在3月龄生长慢个体各组织中的表达均显著高于生长快个体。这些结果表明let-7 miRNAs可能通过负调节其靶基因的表达，从而在团头鲂的生长发育过程中发挥着重要的调节作用。（4）分析了let-7 miRNAs家族10个成员团头鲂性腺发育不同阶段的表达模式，结果显示在不同时期卵巢起主要表达作用的是let-7a/b/d。let-7a/b/d/f/h在Ⅰ至Ⅴ期性腺中的表达量逐步上升，第Ⅵ期显著下降。而垂体中的let-7a/b/e/f在前4期上升，Ⅴ、Ⅵ期下降。这两种表达模式都与鱼类性腺的发育过程吻合，表明let-7 miRNAs可能在性激素及其受体基因两个层次调节性腺发育。（5）GO功能富集分析显示let-7 miRNA靶基因的功能涉及到生长、卵母细胞减数分裂等方面，结合miRanda软件靶基因的预测，推测GHRa、GHRb、MATNa、IGF2基因为let-7 miRNA的靶基因。siRNA转染实验验证了let-7a miRNA对靶基因GHRa的调控作用。表明性早熟个体通过调控let-7 miRNA表达量的升高，调控靶基因GHRa表达量的降低，从而起到抑制团头鲂性早熟个体的生长速度。（6）基于团头鲂选育家系的RAD测序，筛选到与团头鲂生长和性早熟相关联的QTL位点8个。项目研究结果从miRNA水平探讨了鱼类性早熟的分子机制，为团头鲂非性早熟家系的选育提供了分子标记的辅助。