Research On The Role Of MicroRNA164 In The Development And Stress Resistance Of Thellungiella Halophila

Salt stress is an abiotic stress factor seriously affecting plant growth and development which causes serious harm to plants. Salt secretion is one way of plant salt tolerance. Arabidopsis thaliana secrets salt by hydathodes which are located on the margin of leaves. Hydathodes are extremely important in the regulation of water potential and prevence from leaf wilting. MiR164 specifically controls leaf margin development by regulating the expression of CUC2. So the development of leaf serration may affect the development of hydathodes, which even can affect the plant salt resistance ability.MicroRNAs (miRNA) are a family of short endogenous noncoding single-stranded RNA containing about 20-24 nucleotides, they are processed from a single-strand RNA precursor with about 70-80 nucleotides. In plants, these nucleotide RNAs function as negative regulators. MicroRNAs play a sequence-specific negative role in the cleavage or translational repression of mRNA by nearly perfect complementary with target mRNA around the cleavage site, which results in the negative control on the gene expression in the post-transcriptional level. By this way, miRNAs are involved in the biological process, such as growth, development, morphogenesis and the response to the external environmental stress.In 2007, IPS1 (Induced by Phospate Starvation 1) was discovered by Jose′Manuel. It is a non-protein coding gene containing a motif. The motif shows a critical non-perfect mismatch with miR399 (the phosphate (Pi) starvation–induced miRNA) at the expected miR399 cleavage site. Thus IPS1 mRNA sequesters with miR399 which can inhibit miR399 to pair with its target PHO2 mRNA, as a result, the target PHO2 mRNA can not be cleavaged. This mechanism is confined as'target mimicry'which provided a new method for the research of miRNAs.Thellungiella halophila, a close relative of Arabidopsis, is a new salt tolerance model plant which can withstand dramatic salinity stress. It has excellent genetic features such as small genome size, short life cycle, abundant seed and can easily be transformed. So T. halophila was used as the research system in this paper. In the first stage, we cloned ThCUC2 of T. halophile. Firstly, we designed degenerate PCR primers according to the deduced amino acid sequences of CUC2. And the middle sequence of ThCUC2 was amplified. Then we amplified two ends of ThCUC2 respectively. At last, we cloned about 1094 bp ORF of ThCUC2 for a 364 amino acid peptide. The amino acid sequence of ThCUC2 was 86% identical to AtCUC2.In order to verify the expression pattern of CUC2 in T. halophila, we extracted total RNA from roots, stems, leaves, flowers and siliques and analyzed ThCUC2 expression in all these tissuses by Real-time PCR. The results indicated that ThCUC2 expression level was highest in leaves, which is the same as in Arabidopsis.Furthermore, we cloned miR164 and IPS1 genes from the Arabidopsis genome. Then we constructed the full length of miR164 and IPS1 gene into the plant expression vector pCAMBIA-3301H respectively and transferred into Agrobacterium GV3101. Finally, the flower of T.halophila have been transformed with Agrobacterium GV3101 containing the target fragment through floral dip method. The transformed seeds have been screened by using 0.2% herbicide. And we have gained about 17 basta-resistance lines of the gene overexpression of miR164 and 15 basta-resistance lines of the gene silencing of miR164 respectively. Compared with wild type T. halophile, miR164 silencing lines has different phenotype including obvious serration at the margin of leaves, clustered rosette leaves, stem leaves with axillary buds and late flowering time . But there is not great difference in the leave serreation between miR164 overexpression lines and wild type. Early flowering and more branches is the most obvious phenotype of miR164 overexpression lines.