Research On Function Of MiR164 In Thellungiella Salsuginea

MicroRNAs (miRNAs) are a class of endogenous noncoding single-stranded RNA with about 22-23 nucleotides length which are widely distributed in genome of Eukaryotic cells. microRNAs are one of the largest gene familise with the proportion of about 1% in genome which locad in independent locus. The first gene recognized to encode microRNA, lin-4 of C. elegans, were identified on the basis of the developmental timing defects associated with loss-of-function mutations by Lee and Wightman in 1993. It is now known that there are hundreds of microRNAs genes in arabidopsis ,Oryza Sativa, Zea mays, Sorghum bicolo, Saccharum officenarum L and bryophyte. In plants, these nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNA. microRNAs function as sequence-specific negative regulators in post-transcriptional gene silencing by almost perfect complementarity with target mRNA around the cleavage site , which leads to mRNA cleavage or translational repression.By this way,miRNA function in Biological process,such as morphogenesis,growth and development,hormone secretion, signal transduction and the response capacity to the external environmental stress.miR164 is encoded by MIR164A, MIR164B, and MIR164 C,whose targets are six members of the NAC family of transcription factors.miR164 function in auxin Auxin gradients ,the oritation of the Lateral root and the quantity of petal. CUC2 and CUC1 are required for embryonic shoot meristem formation and specification of the organ boundary.which are specificly regulated by miR164a. The mutations in the MIR164A gene deepen serration of the leaf margin. By contrast, leaves of plants overexpressing miR164 have smooth margins. Enhanced leaf serration was observed following the expression of an miR164-resistant CUC2 but not of an miR164-resistant CUC1. Furthermore, CUC2 inactivation abolished serration in mir164a mutants and the wild type. CUC2 and MIR164A are transcribed in overlapping domains at the margins of young leaf primordia, with transcription gradually restricted to the sinus, where the leaf margins become serrated. CUC2 specifically controls leaf margin development under the regulation of miR164.IPS1(INDUCED BY PHOSPHATE STARVATION1) is a non–protein coding gene which is dicovered in the research of miR399 by Jose′Manuel Franco-Zorrilla1 et al. in 2007.It contains a motif with sequence complementarity to the phosphate (Pi) starvation–induced miRNA--miR-399.The pairing is interrupted by a mismatched loop at the expected miR399 cleavage site. IPS1 mRNA is not cleaved but instead sequesters miR-399.IPS1 is a more efficient competitor for miR-399 compared with a cleavable substrate Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. We coin the term'target mimicry'to define this mechanism of inhibition of miRNA activity. Target mimicry provide a new method for miRNA research。Thellungiella salsuginea is close relative to Arabidopsis which is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high seed number and an efficient transformation method. However, T. salsuginea is able to withstand dramatic salinity shock.We cloned IPS1 and miR164 form the genome of Arabidopsis.On one hand,we completed the construction of silencing vector with miR164,on the other hand,we constructed overexpressing vector by partly replacing the sequence of IPS1 with the sequence which is complementary to miR164.In order to check whether CUC2 is also the target gene of miR164 in T. salsuginea,we cloned the CUC2 Homologous gene in T. salsuginea by using Degenerate PCR and 3' -race.