Analysis Of Differential Microrna Expression In Arabidopsis Thaliana Under Heat Stress

The greenhouse effect, which increases the Earth's average atmospheric temperature, inevitably cause a variety of influence on the growth of the plants. Thus, temperature becomes an important factor that affects plant physiology. The enhancement of thermotolerance for plants become a problem that have to be solved imminently.MicroRNAs (miRNAs) are a growing class of small, noncoding RNAs that is encoded by endogenous gene (an average of22nucleotides), they can regulate target gene expression at posttranscriptional regulation. MicroRNA is receiveing the attention of the scientists, it not only can accurately control gene expression at posttranscriptional regulation, but also play an important role when the plant response to various adverse environmental stresses.This study comparing different method of total RNA extraction.Different kinds of arabidopsis heat stress related microRNA screening and the expression differences situation of the stress related microRNA. The specific results are as follows:1. Heat stress related microRNA of bioinformatics analysisThis study through the database looking for arabidopsis heat related microRNA, and using online tools forecast the heat stress response microRNAs target genes. Pre-miRNA should have typical stem-loop secondary structure, and it cannot have big ring and bulge. Mature microRNA should be at one arm of the stem-loop, and it's expression can be detected. In arabidopsis, screening four microRNA (ath-MIR414, ath-MIR415, ath-MIR837-5p, ath-MIR10564-akr) to be involved in response to heat stress through the online database.2. Three kinds of total RNA extraction method of comparative analysisThis study was using modified CTAB method,modified hot-phenol method and Trizol method extraction method to separately extraced arabidopsis total RNA. Compared the effect of three extraction methods, the results show modified CTAB method and modified hot-phenol method, without the high quality and integrity good total RNA, and the total RNA precipitate has some polyphenol compounds, or it is the degradation seriously. The trizol extraction method, it reserves the high quality and integrity of the extrated total RNA. It was the most suitable for follow-up research and experiment.3,RT-PCR detection the four heat related miRNAAccording to the four heat stress related miRNA sequence, this study design specific stem-loop primer for RT-PCR. Detection the corresponding four heat stress related miRNA exists in arabidopsis.4,RT-PCR detection the expression of the four heat stress related microRNA different in two kinds of arabidopsis.Added the heat stress related microRNA for poly(A) tail in the reverse transcription reaction, then only has one step synthesis cDNA. According to One Step PrimeScript miRNA cDNA Synthesis Kit principle and design corresponding miRNA Specific Forward Primer. In the different kinds of arabidopsis, RT-PCR detection the expression of the four heat stress related microRNA under different time of heat treatment.The result reveals the BBX19gene expression through excessive heat stress related plant gene expression level to be higher than wild type plant.