斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因的时序性表达

Temporal expression of miR-9,miR-219 and Dicer gene during zebrafish embryonic development

目的探讨斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因时序性表达规律。方法取正常发育至受精后4、8、12、16、20、24、36、48和72 h(hpf)斑马鱼胚胎各约40枚,用Trizol法分别提取总RNA,茎环法反转录为c DNA,用Real-time PCR检测胚胎miR-9、miR-219及Dicer表达量。结果 miR-9、miR-219及Dicer表达水平在胚胎发育过程中呈现峰谷变化,不同发育阶段之间总的差异有统计学意义(P<0.05)。与胚胎发育全程的基因平均表达水平相比,miR-9在8 hpf之前表达量较低(P<0.05),36 hpf后表达量持续增高,48 hpf和72 hpf时表达量显著高于平均水平(P<0.01)。miR-219在8 hpf之前表达量也处于较低水平(P<0.05),20 hpf后迅速增高,24hpf时达到峰值,并持续到48 hpf(P<0.01)。Dicer在胚胎早期(4 hpf)就有明显表达(P<0.05),随后表达水平迅速下降,16 hpf时达到低点(P<0.05),然后逐渐回升,直至胚胎末期。结论斑马鱼胚胎发育过程miR-9、miR-219及Dicer时序性表达有一定规律,miR-9主要在胚胎后期表达,miR-219主要在胚胎中期表达,Dicer主要在胚胎早期表达,其次在胚胎后期,提示miR-9、miR-219及Dicer参与胚胎发育调控过程。

Objective To study the temporal expressions of miR-9,miR-219 and Dicer gene during zebrafish embryonic development. Methods A total of 40 zebrafish embryos were selected at the 4th,8th,12 th,16th,20 th,24th,36 th,48th and 72 th hour post fertilization（ hpf）,respectively. The total RNA was extracted with Trizol method and was reversely transcripted to c DNA with stem-loop primers. The gene expression levels of miR-9,miR-219 and Dicer were measured by Real-time PCR. Results The temporal mRNA expressions of miR-9,miR-219 and Dicer varied significantly during embryonic development with a pattern of peak curve（ P 〈 0. 05）. Compared with the total average level of gene expression,miR-9 was at a low level before 8 hpf（ P 〈 0. 05）,the expression increased continuously after 36 hpf. The expression levels at 48 hpf and 72 hpf were significantly higher than the total average level（ P 〈 0. 01）. The expression of miR-219 was at a low level before 8 hpf（ P 〈 0. 05）,increased rapidly at 20 hpf,reached the peak at 24 hpf,and maintained until 48 hpf（ P 〈 0. 01）. The Dicer gene in the early stage of embryonic development（ 4 hpf） expressed obviously（ P 〈 0. 05）,then the expression level decreased rapidly,reaching the lowest level at 16 hpf（ P 〈 0. 05）,and then pick up gradually until the last stage of embryonic development. Conclusion There is a certain regularity about the temporal expressions of miR-9,miR-219 and Dicer gene in zebrafish embryonic development. miR-9 mainly expresses in the late stage of embryonic development,miR-219 mainly expresses in the mid-embryonic stage,and Dicer gene mainly expresses in the early embryo and secondly in late embryonic stage. The result indicate that miR-9,miR-219 and Dicer play regulating roles during embryonic development.