| MicroRNAs (miRNAs) are a class of negative regulatory factors which are about-22nt small molecule non-coding RNAs and exist widely in plant genome which play a crucial role in regulating eukaryotic genes. MiRNA involved in almost all important biological processes which functioning mainly by combining to specific target genes to inhibit the products of target proteins or degradation of target mRNAs, thereby regulating gene expression. Consequently, analyzing functions of target genes is an effective method to understand the mechanism of miRNAs and their functions. As a typical tropical energy crop, cassava, genetic improvement of cassava drought resistance could be an important foundation research of the molecular mechanisms under water stress condition. Isolating from drought-related miRNAs and their target genes may open new avenues for genetic improvement of cassava.In this study, expression differences of29drought-related miRNA and33target genes are analysed in the normal growth and under water stress of the cassava varieties SCI24and KU50by real-time quantitative PCR (quantitative Real Time-PCR), besides miRNA target gene are predicted. Furthermore, improved5’RACE are selected to verify their correctness. By cloning and sequecing5’RACE technology-specific PCR amplified products, verify whether these target genes experienced directional cleavage by their miRNAs, and to determine the shear event occurrencd in which nucleartide position of its target genes. The results are as follows:1.Under conditions of drought stress:the ability of drought-resistance of SC124were significantly larger than KU50and their performances were as followings1) under drought stress, leaves of SC124could maintain a certain water potential instead of wilting, the critical point from the bottom of the start wilting was more slowly;2) After rehydration, top leaves of SC124could quickly firm up while KU50were remaining in wilting state, in addition, the SC124issued new roots while the root system of KU50were still in the dry-like.2. Aanalysis of expression level of29drought candidate miRNAs showed that:the number of up-regulated expression miRNA and down-regulated expression were the same in SCI24, while the number of up-regulated expression miRNA were4more times of than in down-regulated miRNAs, which fully proved that the miRNA expression differences between the varieties is very significant. Besides, the number of miRNA up-regulated expression of the two varieties in the three treatments was far more than that in down-regulated. The number of DH, and DNH processing, number of up-regulated miRNA was two-fold more than that in down-regulated miRNA3. We found in the prediction of targent genes of miRNAs that expressed in specific species or treatments (for example:novel-44a.2,171d.2and so on) and the functional annotations of target genes are acetyl-CoA, transcription factors, and hormones and so on. We speculated they may play a vital role in response to drought stress, signal transduction and functional gene expression.4. This study identified cleavage sites of tasiARF-1whose target gene is the auxin receptor factor, which provides important evidence for the further study of the role of target genes in the mechanisms of drought-resisting and its fine regulaiong mechanism mediated by miRNA. |
