草莓microRNA的RT-PCR鉴定

 【目的】microRNAs（miRNAs）在植物的生长发育过程中起着重要作用，本研究旨在建立一种快速准确地鉴定草莓中保守miRNA的方法。【方法】以草莓叶片为试材，在利用改进的CTAB法成功富集小于150 bp的小分子RNA的基础之上，采用3′端加接头、5′端和3′端两端加接头、利用茎环等3种RT-PCR策略来检测鉴定草莓中保守miRNA，同时比较总核酸、总RNA和小分子RNA等不同种类的模板对利用茎环RT-PCR检测miRNA的影响。【结果】对于植物中20种miRNAs，通过3′端加接头方式鉴定出7种;通过两端加接头方式鉴定出1种;而利用茎环的方式鉴定出15种。分别以3种核酸为模板的茎环RT-PCR的扩增效果没有差异。【结论】总核酸的提取是最快速最简单的，因此，以总核酸为模板，利用茎环RT-PCR是鉴定植物中miRNA的最简便快速而有效的方法，这种方法的可行性已在苹果、葡萄等植物上得到验证。

Identification of MicroRNA in Strawberry by RT-PCR

【Objective】 microRNAs (miRNAs) play an important role in plant growth and development. The aim of this paper is to develop a quick method for exact identification of the conserved miRNAs from plants. 【Method】 High quality small RNAs (less than 150 bases) were isolated from strawberry leaves with the modified CTAB method. Three kinds of RT-PCR methods, using 3′ adaptor, using 5′ and 3′ adaptors and stem-loop primers, were compared. Three kinds of templates, total nucleic acid, total RNA and small RNAs were compared in identification of miRNAs with stem-loop primers. 【Result】 For twenty miRNAs of plants, seven miRNAs were identified from strawberry plants by using 3′ adaptor, only one miRNA was successfully identified with the method of 5′ adaptor and 3′ adaptor, while fifteen miRNAs were identified by using stem-loop primers. No difference was found in PCR fragments by using three kinds of nucleic acids as templates. 【Conclusion】 Because the extraction of total nucleic acid was very simple and rapid, the suitable method for identification of the conserved miRNAs from plants with RT-PCR was to use stem-loop primers and total nucleic acid as template. This method had been approved on other plants, such as apple and grape.