Differential Expression Of MicroRNA In Response To Drought And Salt Stress In Mulberry

MicroRNAs( miRNAs) are 18-24 nucleotides long sequences of endogenous non-coding RNAs, they exist extensively in animals, plants, bacteria, viruses. MiRNAs have been dicovered to regulate the expressions of genes by target cleavage and by inhibiting mRNA translation in organisms, miRNAs interfer in the growth process and the abilities to resistant against biolic and abiolic stress.Mulberry is one of the most important economic crops, but various abiotic stress conditions and plant diseases or insect pests account for the severe bad crop of mulberry.The study of the mulbrry miRNAs expression in response to stress plays an important role in the protection of mulberry germplasm resources and increasing productions. Compared with other crops, the study of mulberry miRNAs is still at the initial stage, few mulberry miRNAs is known. Therefore, a research on the mulberry microRNAs in mulberry resources development is a very meaningful work. Drought and salt treated mulberry were prepared for studying the expression of related miRNAs under drought and salt stress. The results are as follows.1.Mulberry saplings were treated by drought and salt(0.3M NaCl) stress. Spires from drought treatment, salinity treatment and control treatment were made as research object. 3small RNA libraries were constructed and deep sequenced by Solexa sequencing platform.13878088, 13619220, 15711033 high quality reads were sequenced and 50 percent were perfect matched onto the Morus notabilis genome. 69 conserved miRNAs and 205 Novel miRNAs were predicted by the analysis of the sequening data. 45 percent of the miRNAs were 24 nt in length.2.The expression study was performed between the miRNAs in prediction, and 20 of them displayed significant differential expression between drought and salt treated samples compared to the control. Under drought stress, 13 miRNAs were significant differential expressed, 4 of them were up-regulated, and 9 miRNAs were down regulated. While under salt stress, 12 miRNAs were significant differential expressed, 4 of them were up-regulated,and 8 were down regulated. Then stem-loop RT-PCR method was used to verify the expression of the 20 miRNAs. As a result, 4 miRNAs were up-regulated and 9 were down regulated under drought stress, the trend was the same as the result from the sequencing data.However, 7 miRNAs increased the expression and 5 miRNAs decreased the expression inrespond to salt stress.3.By using bioinformatics online software psRNAtarget and referencing genome of Morus notabili, targets of the 274 miRNAs were predicted. Finally, 577 target genes were obtained from 175 miRNAs. To confirm the way miRNAs regulate their targets, miR395 a was selected as an example. The target of miR395 a was ATP sulfurylase 1 gene and was down-regulated in respond to drought. In this study, real-time RT-PCR was applied to the quantification of the target. Up-regulated expression of this gene was observed during different period of the stress treatment, proving the way miR395 a function on the target was on post transcriptional level. To verify the target of miR395 a, RLM-5’RACE was conducted.After adaptor connecting, inverse transcription, specific amplification and sequencing, it was found mi R395 a cut the target between the 10 th and 11 th nucleic acid bases in the combination area.