龙眼miR403及其候选靶标对外源激素的响应模式以及在龙眼体胚中的表达模式

The response patterns of miR403 and its candidate targets to exogenous hormones and their expression profiles in the longan somatic embryo

MicroRNA403(miR403)为双子叶植物特有的miRNA家族,其在双子叶植物抗病、逆境胁迫和生长发育中具有重要作用.为了解miR403及其候选靶标对外源激素的响应模式以及在龙眼体胚发生过程中的表达模式,利用psRNAtarget对miR403潜在靶标进行预测,采用改良RLM-RACE技术验证其裂解位点,通过qPCR技术检测miR403及其候选靶标对外源激素的应答及在龙眼体胚中的表达模式.结果显示,共获得41个龙眼miR403的潜在靶标,包含4个PPR(Pentatricopeptide repeat protein)和3个PCNT115(Auxin-induced protein PCNT115),却未发现AGO2(Argonaute2).4个PPR中Dlo014588.1和Dlo014589.1序列一致,而3个PCNT115基因序列在所预测的miR403结合位点上下游序列基本一致.裂解位点显示,miR403不具备裂解AGO2 mRNA的能力,但介导PPR(Dlo014588.1)和PCNT115 mRNA的裂解,裂解位点位于miR403 5′端的第2和第3个碱基之间. qPCR显示,miR403响应脱落酸(ABA)信号并显著上调,PPR和PCNT115对不同浓度的ABA呈现不同的表达模式,PPR和PCNT115在5μg/L ABA时显著上调,随后,PPR先显著下调(50μg/L ABA),而后显著上调(5 000μg/L ABA),而PCNT115呈显著下调趋势;miR403不响应赤霉素(GA3)信号,而PPR和PCNT115随着GA3浓度升高而上调;miR403可以响应不同浓度的水杨酸(SA)和2,4-D信号显著下调,而其靶基因显著上调.不同胚性培养物中qPCR显示,miR403仅在龙眼胚性愈伤组织(EC)高度表达且在龙眼体胚发生过程中显著下调,PPR在EC和子叶胚(CE)高度表达,PCNT115在CE和成熟胚(ME)高水平表达,三者之间并未呈现明显的负调控趋势.本研究表明在龙眼体胚中miR403并不靶向调控AGO2,而是调控PPR和PCNT115;另外,miR403可能响应ABA、SA和2,4-D调控PPR和PCNT15参与到龙眼体胚发生过程中.

MicroRNA403(miR403) is a miRNA family unique to dicotyledons that plays vital roles in antiviral defense, stress resistance, and growth and development of dicotyledons. To understand the response patterns of miR403 and its candidate targets on exogenous hormones and their expression profiles in the longan somatic embryo, the candidate targets of longan miR403 were predicted using psRNAtarget software and the cleavage site of candidate targets were verified by the modified RLM-RACE(RNA ligase-mediated amplification of cDNA ends);moreover, quantitative real time-PCR(qPCR) was performed to identify the expression profiles of miR403 and its targets in longan friable-embryogenic callus(EC) treated with different hormones and longan somatic embryos at different stages of development. A total of 41 candidate target genes of longan miR403 were predicted. Among these, 21 were annotated in NCBI, including four pentatricopeptide repeat proteina(PPR) and three auxin-induced proteins PCNT115(PCNT115). Multiple alignment of PPRs and PCNT115 s revealed that Dlo014588.1 and Dlo014589.1 sequences were identical, and PCNT115 s were basically identical upstream and downstream of the miR403 binding site. The cleavage site of Argonaute 2(AGO2) suggested that longan miR403 cannot cleave AGO2 mRNA. The cleavage sites of PPR(Dlo014588.1) and PCNT115 were located between the 2 th and 3 th base at the miR403 5’ end. After different concentrations of abscisic acid(ABA) were used, miR403 was significant upregulated. PPR was significantly upregulated upon 5 and 5 000 μg/L ABA treatment and significantly downregulated upon 50 μg/L ABA treatment. PCNT115 was significantly upregulated after 5 μg/L ABA treatment and significant downregulated when the ABA concentration was increased. miR403 did not respond to GA3, but GA3 upregulated the expression of PPR and PCNT115. Salicylic acid(SA) and 2,4-D downregulated the level of miR403, and the expression trends of PPR and PCNT115 showed a negative regulator pattern with miR403. The results of qPCR in the longan somatic embryo at different stages indicated that miR403 was highly expressed only in EC, PPR was highly expressed in EC and cotyledonary embryos(CE), and PCNT115 was highly expressed in CE and the mature embryo(ME). There was no clear negative regulation trend between miR403 and its targets. The results showed that miR403 could not cleave AGO2 in the longan somatic embryo but regulated PPR and PCNT115 at the transcription level. In addition, miR403 might response to ABA, SA, and 2,4-D signals, and miR403, PPR and PCNT115 were involved in the morphological development of the longan somatic embryo.