麻竹miR172a靶基因DlAP2的克隆及其表达

Cloning and Expression Analysis of miR172a Targeted Gene DlAP2 in Dendrocalamus latiflorus

摘要:

为了解开花麻竹(Dendrocalamus latiflorus)的DlAP2 基因功能,采用RT-PCR 和RACE 技术克隆了miR172a 靶基因AP2同源序列cDNA 全长,命名为DlAP2.结果表明,DlAP2 基因cDNA 全长为1729 bp,包含5' 端非编码区81 bp、开放阅读框1464 bp、3' 端非编码区160 bp 和24 个碱基的Poly A 尾巴,在编码框靠近3' 端130 bp 处有1 个高度匹配miR172a 的结合位点(CTGCAGCATCATCAGGATTCT).DlAP2 编码487 个氨基酸的蛋白,具有两个AP2 结构域,属于AP2/ERF 家族AP2 亚家族的AP2 组,与来自其它单子叶植物的AP2 蛋白均有较高同源性.RLM-5' RACE 分析表明,miR172a 主要在靶序列的第11~12 个碱基之间剪切靶基因DlAP2 的miRNA.qRT-PCR 结果表明,麻竹花芽中DlAP2 基因的表达规律与miR172a 表达变化正好相反,证明miR172a 对DlAP2 基因的表达具有调控作用.

Abstract:

In order to understand the function of DlAP2 in Dendrocalamus latiflorus, one miR172a targeted gene named as DlAP2 was cloned from D. latiflorus by RT-PCR and RACE. Sequence analysis showed that the full length cDNA of DlAP2 was 1729 bp, including 5' untranslated region (UTR) 81 bp, open reading frame (ORF) 1464 bp, 3' UTR 351 bp, 24 bp polyA, and one miR172a complementary site (CTGCAGCATCATCAGGATTCT) at 130 bp of the 3' end in ORF. DlAP2 encodes a putative protein with 487 amino acids with two AP2 domains, which indicate that it belongs to AP2 group of AP2 subfamily in AP2/ERF family. DlAP2 has a high homology with those AP2 from other monocots. RLM-5' RACE analysis showed that DlAP2 was regulated by miR172a through cutting mainly at the site between the 11^th and 12^th bases. Real-time quantitative PCR results showed that the expression pattern of DlAP2 was opposite to that of miR172a in flower buds. These validated that miR172a played a regulatory role in regulating the expression of DlAP2.