| Dongnongdongmai1is the uniqueness cultivated wheat variety which can survive in Heilongjiang province (overwinter survival rate more than85%), and it has important cold resistant miRNA and relevant resources of target genes. This study is aimed at exploring the important stress resistant miRNA resources of Dongnongdongmai1. On one hand, it will reveal strong cold resistance mechanism of Dongnongdongmai1, widen the cold resistant hereditary basis of wheat, provide useful traits and sources of cold resistant miRNA genes to wheat genetically modified cultivation, and make an important contribution to the improvement of cultivation in winter wheat in the north of China. On the other hand, it will also supply important information for the deep research of miRNA metabolism.In this study, tillering nodes of Dongnongdongmai1in three different growing periods were adopted as materials, depth sequencing was conducted through HiSeq technology in three different growing periods, the sequencing results were statistically analysed, then the sequencing fragments were screened by Rfam(10.1), Genbank database, and repeat sequence, finally, all the fragments were classified, and all miRNAs were found in three different period including known and unknown miRNA. Known miRNAs were analysed, first of all, base bias analysis was adopted, and miRNA expression profiling was build in three temperature points, then different analysis was conduced in miRNA, meanwhile, miRNA differential expression profiling was build. Differential expression analysis and family analysis were adopted on known miRNA, and unknown miRNAs were predicted and analyzed in including base bias analysis, secondary structure prediction, unknown miRNA differential expression analysis and target gene prediction and functions prediction. The results are as follows:1. Wheat Small RNA sequencing data processing and Statistics Analysis The largest numbers of the small RNA we have got were24nt and21nt among the fragments of the length between10nt and35nt by adopting Solexa Sequencing. At last, clean reads accounts (10024183,9448982,10868683) were obtained at3different temperature points.The possible rRNA, scRNA, snoRNA, snRNA, tRNA from reads fragments were erased by the comparison between Rfam(10.1) and Genbanak database. The rest of the Different respective sequences were erased by repetitive sequences comparison, at last, sRNA categories and selection were conducted, the total accounts we selected at3different temperatures of miRNA are1299843,1167152,1544325.2. Identification and Difference Expressions of the Known miRNA under Cold InductionThe known miRNA expression profile was built.of the wheat. There are44known miRNA in wheat miRBase, but35,29and31known miRNA in each3samples (tillering nodes under5℃、-15℃and-25℃) were found in Dongnongdongmai1. The accounts of wheat conservative reading frame were analysed, and a big difference was found in express frequency of miRNA. Among them, the higher frequency of11miRNA was found,5miRNAs were referring to cold inducement in the difference analysis, they are tae-miR156,tae-miR398, tae-miR160and tae-miR444and tae-miR444b, and tae miR156expression was in the highest abundance, it had a downregulated trend, and tae-miR398was significantly up-regulated expressed, while tae-miR.160taemiR444and tae-miR444b significantly were significantly down-regulated expressed. Based on the difference analysis and reported target genetic analysis, we infer that above5miRNA have something to do with the cold resistance of Dongnongdongmai1.3. The Identification of a Part of Known miRNA and Difference Analysis of Dongnongdongmai1567known miRNA were found in Dongnongdongmai1under the cold induction, among them,164known miRNA were in OEG analysis. Among the selected14known miRNA in OEG analysis,3miRNAs were connected with flower development, they are Tae-miR168, Tae-miR166, Tae-miR172.5miRNAs were connected with cold induction, they are Tae-miR169, Tae-miR397, Tae-miR319, Tae-miR165,6unknown were from cold induction and significant in OEG known miRNA. They are Tae-miR5565,Tae-miR5029, Tae-miR5070、Tae-miR5139, Tae-miR5218,Tae-miR3704.4. Predicting the New miRNA378new wheat miRNAs were discovered on the three low temperature points in this study. The number of the new wheat miRNA on each low temperature point are124,96and125, and the predicted accounts of target genes are89,78and99. Among the predicted miRNA database, fully expressed and significant in OEG analysis ones were choosed. Finally,14new miRNA among "Dongnongdongmai1" were choosed and new miRNA expression profile was built and predicted, take advantage of the Mineap software,14miRNAs were predicted which had typical secondary stem-loop structure. Furthermore, the selected14new miRNA were predicted in target genes, seven of them were predicted in target genes, while another seven did not. In this study, the3interesting new miRNAs were analysed, the function of the3new miRNA was predicted by analysing the target genes. |
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