| MicroRNAs(miRNAs) are 21~24 nt endogenous RNAs that are highly conserved among higher plants. MicroRNAs use base-pairing interactions to regulate mRNA expression by cleaving the targeted transcript, or translational inhibition following imperfect pairing. miRNAs can effectively control the expression of target genes, so carry out the research on miRNA level can explain the relevant expression mechanism in essence. Moreover, owing to miRNA is highly conserved in the evolution of higher plants, the research on which may seem more universal. Numerous recent studies have shown that miRNAs and their targets play important roles in response to abiotic stress. Drought is one of the most common environmental stresses that threatens agroforestry and results in deterioration of the environment. According to reports, there are up to 500 million kilogram crop yield reduced by drought every year in China. Therefore, more effective xerophytic breeding strategies are urgently required to improve plant survival under extremely harsh environments. Caragana intermedia, of the Leguminosae family, is a deciduous, perennial shrub that is widespread in the sandy grassland and desert regions of northwest China and Mongolia. It has important ecological and economic value in this area, including drought resistance, sand-fixation, water and soil conservation, as well as a nutritious pasture. Because of its strong adaptability and stress tolerance, C. intermedia is an ideal candidate plant for studying the mechanisms of drought tolerance and creating drought-resistant forest in China.Previous studies have demonstrated that mi R2118 is closely related to drought response in plants. However, its moderating effect on the drought has not been confirmed in any plant. In this study, we predicted two potential target genes of miR2118 from the C. intermedia transcriptome and cloned them. We also characterized the expression patterns of mi R2118 and its target genes under drought stress in various tissues of C. intermedia. The main results are as follows:(1) Two target genes of ci-miR2118 were predicted and cloned in Caragana intermedia and then designated as CiDR1 and Ci DR2. Bioinformatics analysis show that they belonging to TIR disease resistance protein family and all contain 3 conserved domains TIR, NBS and LRR. But the two genes located at different organelle, which may cause far phylogenetic relationships. The occurrence of mi R2118 target sequence in both genes indicated that they might be targets of mi R2118.(2) qRT-PCR results showed that miR2118 was co-expression with its two target genes in C. intermedia in infancy period, and exerted reverse expression patterns with CiDR1 in adulthood. We therefore speculated that miR2118 regulate its target gene CiDR1 by phase-specific regulation, which means it is only functioned in mature period and non-function in juvenile period. The CiDR2 gene may controlled by multiple genes on the growth and development stage.(3) The expression pattern of CiDRs in C. intermedia under drought stress was investigated using qRT-PCR. CiDR mRNAs were expressed at high levels in all organs of untreated plants. CiDR1 was negatively correlated with corresponding miR2118 expression at most phases under drought treatment. While the expression of CiDR2 was positively correlated with ci-miR2118 at all time-points. The target gene CiDR2 may function as a “tuning targets” of miR2118, for which mi RNA regulation adjusts the protein output in a fashion that permits customized expression in different cells types while achieving a more uniform expression level within each cell type.(4) Overexpression miR2118 tobaccos exhibited higher miR2118 expression in their roots, stems, and leaves than wild-type plants, the highest expression presented in stems reach to 150 times than wild-types. Our studies strongly indicate that the transgenic tobacco plants possessed higher water retention ability than wild-type, and that ci-miR2118 functioned as a positive regulator in responding to drought stress.(5) Overexpressing ci-miR2118 also caused severe growth abnormalities in tobacco, which exhibited stronger stems, shorter but thicker roots, and an absence of root hairs in the maturation zone. Micro-Structure analysis showed that xylem cells in transgenic lines were larger and more numerous than in wild-type tobaccos, thus occupying more space between the central and peripheral zones. The increase of vessel number per unit area can provide guarantee for transportation system, improve the effectiveness and safety of water transportation, also can reinforce the ability to bracing. This change in morphology exhibited distinctly xeromorphic characteristics. We can assume that ci-miR2118 participates in the regulation of normal development in Tobacco.In conclusion, miR2118 and its target gene adjusting their expression to help plants adapted to drought, so to speak, they play an important role in drought stress responses. Overexpression of ci-mi R2118 can not only enhance the drought resistance to plants but also change the phenotype of plants. We assume that it may also relate to the growth and development in plants. |
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