| MicroRNAs(miRNAs) are important factors involved in plant growth and development of gene expression regulation. Most miRNAs target gene encode regulatory proteins, indicating miRNAs are the main regulatory factors in plant and they are in the center of gene expression and regulation. MiRNAs of various families were expressed significantly different, in specific tissue or in specific developmental stages. And they regulated target gene at different levels (transcriptional, posttranscriptional, translation, etc.). Tobacco is an important economic crop. Topping is an important agronomic technique in tobacco planting. The reason of enhancement of nicotine synthesis was focus point to tobacco researchers. To study the regulation of nicotine synthesis ability in roots after topping, miRNAs in the root tips before and after topping of tobacco were deep sequenced and the expression profilings were obtained. In this article, the expression profilings were analyzed with bioinformatics methods, and the key enzyme of nitrogen metabolism, GS2, was analyzed, too. The results were showed as follows:1. The sequences were annotated and got its expression information. Through common sequence analysis of the samples, it shows the characteristics of tobacco smallRNA and its expression variation. Three peaks of smallRNA length were found before and after topping respectively, in the 21nt 22nt and 24nt position, which is consistent with current studying. After topping, the number of smallRNA less than 20 nucleotides is more than the before topping ones. However, before topping the length greater than or equal to 20 is more than the number of nucleotide smallRNA after topping. And before topping, the number of 24nt small RNA is 30% higher than smallRNA after topping. In the total sample sequences the percentage of common sequences is 60.96%, but changed into 9.56% in non-redundant distribution, indicating that the common small RNA control the normal growth and development of tobacco. rRNA and tRNA is in the overwhelming place by 99% of non-coding small RNA besides miRNA, and snRNA and snoRNA only take 1% whatever before and after topping. Before topping,the quantity of rRNA were more than the quantity of tRNA, but the results shows conversely after topping.This phenomenon shows that may be rRNA, tRNA were involved in the steps of protein synthesis.2. The numbers of the smallRNA whom exactly matched MiRBase were 246 ;after topping,there are 8 miRNAs which expression was significantly increased 20 times higher than themselves before topping;there are 7 miRNAs expression was less than 25% before topping. After topping there were 9 miRNAs which were no significant change; there were 4 specific miRNAs before topping and there were also 4 specific miRNAs after topping. Using bioinformatics methods we predicted the characteristics of the structure of unannotation smallRNA to search new miRNAs based on miRNA hairpin structure, refolding free energy and enzyme sites,we also analyzed their secondary structure features,we found 56 miRNA molecules are not reported in tobacco.3. We cloned miRNA164 target gene with in in silico cloning: Through online information inquiry, we got the tobacco NAC-like gene cDNA sequence (1348bp) which is the target gene of miRNA164 by in silico cloning. And it was determined to be tobacco NAC transcriptional factor, after RT-PCR and sequencing.4. Predict and analyze the glutamine synthetase (GS) in the different tobacco varieties. We predicted sites of 14-3-3 protein binding sites in noncleaved tobacco GS2 peptide chain near the Ser97 position and it may have a similar way in post-translational regulation with alfalfa.We have predicted the MAPK and PKD phosphorylation sites in tobacco GS2 mature peptide. The Spc24 domain was predicted in GS2 of two kinds of tobacco and Arabidopsis, which indicated that NaGS2 and NsGS2 may be involved in the process of chloroplast development. |
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