豌豆中抗寒相关性miRNAs功能特异性验证及其克隆的研究

Identification and isolation of the cold-resistance related miRNAs in Pisum sativum Linn

以正常(对照)和寒冷诱导豌豆(Pisumsativum Linn)幼苗为研究对象,用已知模式植物拟南芥中抗寒性相关的4种miRNAs序列(miR319、miR393、miR397、miR402)设计引物,通过半定量PCR法,检测在豌豆中正常条件和寒冷诱导下可能出现的相关miRNAs.在此基础上,选取模式植物中已知的miR319序列,设计特异引物,寻找豌豆抗寒性相关的miRNAs.结果表明:miR319、miR393、miR397、miR402均存在于豌豆中,并且冷处理诱导下表达量都有所上升,此结果验证了抗寒性miR家族在植物间存在保守性的特点;特异性寻找克隆、测序结果表明,新的与抗寒性相关的miRNAs也存在于豌豆中.本研究建立的寻找或验证miRNAs的方法是可行的,并为在各种生物中miRNAs功能研究与应用打下良好基础.

Sweet Pea（Pisum sativum Linn） seedlings were cultivated in the normal（as control） and the cold conditions separately.According to cold resistance of the four miRNAs（miR319、miR393、miR397、miR402） which had been found in the model plant—Arabidopsis thaliana,the primers were designed to detect whether the Sweet Pea seedlings which had been treated in cold condition and the normal condition separately exist the related miRNAs or not by using the methods of Semi-Quantitative RT-PCR.Further more,a pair of specific primer was designed by comparing with the sequence of miR319 known the cold resistance in the model plants might also find in Sweet Pea after the specific RT-PCR.The results showed that miR319,miR393,miR397,miR402 probably exist in Sweet Pea,and the level of their expressions were gone up after the Sweet Peas had been treated in cold condition.One thing could be sure that the miR family of cold resistance do have the characteristic of reservation in plant.The new cold-resistance miRNAs were also found in Sweet Pea after the process of specific search,clone and sequencing,the way to find or testify functions-related miRNAs was proved to be feasible.