Screening And Verifying The Cold-stress Responsive MicroRNAs From Dongxiang Wild Rice (Oryza Rufipogon Griff.)

Rice is one of the most important food crops in the world. Now chilling injury is the main factor that influence its yield.So the research on the genetic mechanism of rice on cold tolerance has great significance.More and more studies has shown that microRNA is a kind of non-coding RNA targeting m RNA which lead a role of negatively regulate the broad pathways which involved in the response of stress. As a well know thing in China,Dongxiang wild rice(DXWR) has been so far the most north distributed one and it has a amazingly strong cold tolerance ability.Naturally,DXWR become a germplasm resources for cold resistance.But to date,whether MicroRNA lead a role in the cold tolerance of DXWR is still unclear. In this study,Illumina sequencing technology was used to obtain the microRNA sequences of DXWR, then with the analysis of bioinformatics methods, cold stress relatied microRNAs were filt out.At last,the experiment was carried out to verified the filt result above.It was expected this can lay a foundation for the clear explantion of the strong cold tolerance ability of DXWR.The results are as follows:Analysis of small RNA Library of high-throughput sequencing data15 day old seedlings of Dongxiang wild rice was used as the materials for the contruction of sRNA library by the Illumina high-throughout sequencing technology.The total number of high quality clean reads producted by that was 4940252.Take the sequence of precursor and mature microRNA as the alignment reference,93 conserved microRNAs were found,in which whose reads number ranked in top five were osa-miR159(29637), osa-miR396(15505), osa-miR166(4974), osa-miR156(1771),osa-miR164(1209). Finding out the pre-microRNA sequences in Nipponbare rice genome,under the principle of minum free energy and judged by that if the secquences can form the fold-back secondary structure,other 354 different reads meeted both of the conditions were regarded as the novel microRNA.the bioinformatic processing to all the microRNAs.Each microRNA target gene prediction software was designed according different principle, this will lead to the different prediction results. So the strategy that getting the intersection part of target genes of every microRNA will be more credible. Targetgene prediction softwares used in this thesis were online prediction software psRNAtarget(http://plantgrn.noble.org/psRNATarget/) and localized software Targetfinder.pl.Using the self compiled Perl program,1994 uni-target genes were obtained. Then, GO and KEGG analysis for these genes was performed on the online software blast2 go. The cold relatied genes were filt out through literature access.Finally,145 novel microRNA and 22 coserved microRNA which has a close relation with cold stress was obtained.3. Experiment identification of the bioinformatic processing result with cold stress.Before bioinfomatic screening to all microRNAs,the phenomenon that conserved microRNAs were mush easier to be verified than the novel microRNAs by real-time qPCR in pre-experiment.Considering the microRNAs included in the database(miRbase14.0)were only 713 by so far,the real microRNAs in the 354 novel microRNA must be very rarely.Actually,no novel microRNA was identified but almost all conserved microRNAs were idendified in the pre-experiment. So all the selected microRNAs that were prepared to be verified were conserved ones. Without the help of software,a set of three primers-reverse transcription,right forward,left forward-for each miroRNA were designed artificially for the six random selected conserved microRNAs.The seedlings were treated in 4 ℃ environment and the samples were taken in these time point-0h,1h,3h,6h.The real-time qPCR was used to calculate the amount of microRNAs’ changing with the time.The result was showed with bar chart.In summary,the selected five microRNAs are verified in the Dongxiang wild rice.After the treatment of low temperature,their expression changed but in different trend.It improved that filting the goal microRNA by bioinformatic method and verified by the later experiment is effective.