Pre-miR172及miR172调控油菜AP2基因表达的规律分析

Analysis of expression regulation of AP2 gene by pre-miR172 and miR172 in rape

关键词:  油菜, AP2, miR172, pre-miR172, 表达规律

Key words:  rape, AP2, miR172, pre-miR172, expression pattern

摘要:

为探究油菜miR172前体(pre-miR172)及成熟体(miR172)对AP2基因的调控功能,该研究通过生物信息学方法对miR172和AP2启动子进行调控元件预测,分析6条油菜AP2基因的进化关系及miR172与AP2的靶向关系; 通过qRT-PCR方法检测AP2、miR172和pre-miR172在早熟和晚熟油菜不同组织的表达规律; 比较分析miR172丰度和AP2表达量间的相关关系,以及比较分析 pre-miR172和miR172在表达水平上的相关关系; 通过过表达pre-miR172,再次验证pre-miR172对成熟体miR172及AP2的作用。结果表明:(1)miR172和AP2启动子区均存在调控花发育的顺式元件。(2)6条AP2序列均经历了强烈的纯化选择,均具备miR172的结合位点,属miR172的靶基因。(3)miR172家族成员均可促进早熟油菜AP2表达,但miR172d作用不明显。在晚熟油菜中,miR172a和miR172c作用微弱,miR172b和miR172d二者共同发挥作用降低AP2的表达水平。(4)pre-miR172家族对于早熟油菜中miR172家族的表达水平均有促进作用; 在晚熟油菜中pre-miR172a和pre-miR172b对其成熟序列的形成发挥正调控作用,pre-miR172c和pre-miR172d则对于其成熟序列的形成发挥负调控作用。过表达pre-miR172后,miR172和AP2表达规律与上述结果保持一致,证实pre-miR172对miR172及AP2的调控功能。该研究结果丰富了油菜AP2基因的功能调控路径,为基因的调控功能研究提供了新的思路。

Abstract:

The appropriate flowering time is of great significance for crop yield. In the cropping pattern of “rice-rice-oil” in southern region, researching on the flowering period of Brassica napus can provide a theoretical basis for the breeding of early maturing rape varieties. The AP2 family transcription factors in rape are widely involved in the growth and development and play an important role during flower development. However, there are few studies exploring the regulation of AP2 at the microRNA level. In order to investigate the regulatory functions of the miR172 precursor(pre-miR72)and mature body(miR172)on AP2 gene in rape, the regulatory elements of miR172 and AP2 promoters were predicted based on bioinformatics, then the evolutionary relationship of six rape AP2 genes and the targeting relationship between miR172 and AP2 were analyzed, and the expression patterns of AP2, miR172 and pre-miR172 in different tissues of early and late maturing rape were detected by qRT-PCR. Finally, the correlation between miR172 abundance and AP2 expression level was studied, as well as the correlation between pre-miR172 and miR172. The results were as follows:(1)Both miR172 and AP2 promoter regions had cis-elements that regulated flower development.(2)The six AP2 sequences holded the strong purification selection, and they were the target genes of miR172 based on their binding sites for miR172.(3)miR172 family could promote the flowering of early maturing rape by increasing AP2 expression levels, except for miR172d. In late maturing rape, miR172a and miR172c performed weakly in flowering, while miR172b and miR172d worked together to reduce the expression level of AP2 to inhibit flowering.(4)The pre-miR172 family had a promoting effect on the expression level of miR172 family in early maturing rape; in late maturing rape, pre-miR172a and pre-miR172b exerted positive regulation on the formation of their mature bodies, while pre-miR172c and pre-miR172d exerted the opposite effects. After overexpression of pre-miR172, the expression patterns of miR172 and AP2 remained consistent with the above results, confirming the regulatory function of pre-miR172 on miR172 and AP2. The results of this study enrich the functional regulation pathway of rape AP2 gene, and provide new ideas for the study of gene regulatory function.