Cloning And Functional Analysis Of MIR172 Gene In Doritaenopsis Hybrid

Doritaenopsis hybrid, an intergeneric hybrid of Doritis and Phalaenopsis, is with important economic value. The increasing production cost, resulted from inherited cooltemperature-supplied for floral bud dormancy releasing, has inhibited the sustainable development of the commercial production in Phalaenopsis. microRNAs play an important role in terms of floral induction. microRNA172(miR172) is involved in the regulation of plant flowering by negative control of its target genes. The experiment was designed to explore whether miR172 in Doritaenopsis(DhmiR172) participates floral induction and its regulatory mechanism. In this study, two MIR172 s genes and three candidate target gene fragments from Doritaenopsis were firstly cloned. Secondly, the temporal and spatial expression of miR172 and its relative genes were analyzed by Real-time PCR. Finally, CaMV 35S:: DhMIR172 and CaMV 35S::anti-Dh MIR172 transgenic Arabidopsis thaliana and Nicotiana bacocum assay were applied to detect the putative character and function of this gene. The main results are as follows:(1) Two novel MIR172-like sequences of different lengths were amplified by PCR against genomic DNA in Doritaenopsis hybrid. One fragment was 100 bp in length annotated as DhMIR172-1, the other was 211 bp as DhMIR172-2. Sequence analysis showed that both obtained MIR172 s contained a high conserved region that was denoted as the mature body of miR172. Phylogenetic results showed that the DhMIR172-1 and DhMIR172-2 are closer to ath-MIR172 b, zma- MIR172 e, sbi- MIR172 b and sbi- MIR172 f.(2) Three fragments of DhmiR172 candidate target genes DhAP2, DhTOE1 and DhTOE2 were isolated by RT-PCR against cDNA in Doritaenopsis hybrid. Sequence analysis showed that the three fragments all contained one or two conserved domains denoted as AP2 family, which is the character of target genes to miR172.(3) Real-time Quantitative PCR were conducted to investigate the changes of mRNA transcripts of the miR172 and its target genes in different periods and organs during floral induction in Doritaenopsis hybrid. The results showed that DhmiR172 made greater responses to the cold contrasted to the warm and prominent tissue specificity in Doritaenopsis hybrid; The markedly negative correlation between DhmiR172 and its three candidate target genes were also fund in Doritaenopsis hybrid treated with low temperature. DhmiR172-FT regulation in coldinduced flowering was discovered before and after pedicel extraction, and the mRNA transcripts level of DhmiR172 in leaf of hypothermia group changed together with DhFT gene.(4) Transformation of A. tumefaciens-mediated plants were achieved by a floral dipping method in Arabidopsis thaliana and leaf disc method in Nicotiana bacocum. GUS assay were performed for positive transgenic plants. One antisense transgenic A. thaliana was survived and exhibited with short in height with less apical dominance and a remarkable late flowering phenotype. The real-time qPCR results showed that the endogenous miR172 was down-regulated and the target genes(athAP2, athTOE1, athTOE2) were up-regulated in transgenic A. thaliana. Twelve transgenic N. bacocum were also regenerated, and they were in the stage of vegetative growth at present and still not flowering.