Functional Analysis Of MicroRNA156Controlling The Developmental Phase Transitions In Tobacco

Tobacco is widely planted and processed in China. It is an important economic crop with highplanting area and yield all over the world. Along with the development of molecular biology andgenomics, modern biotechnology has begun to widely used in improving the hereditary character ofmodel plant tobacco and many other crops.MicroRNA156(miR156) is highly conserved in all land plants, it controls a series of SPL genesthrough a complex gene regulation network, such as post-transcriptional degradation and translationalinhibition to regulate plant growth and development, which provides an endogenous age cue fordevelopmental timing in plants.In this study, nta-miR156c analyzed by phylogenetic tree was selected for investigation.Overexpressing vectors contained the sequence of nta-miR156c precursor and inactivation of miR156with a target site mimic were introduced into Nicotiana tabacum L. cv. NC89to investigate the effect ofmiR156in tobacco. We detected the morphology which can be used to distinguish the phase change intobacco vegetative stage. With the support data of physiology and the expression of downstream genes,the molecular mechanism of vegetative phase transition in tobacco has been roughly revealed in thisstudy.The main results are as follows:1.Phylogenetic tree showed that tobacco miR156c gene has high homology with ArabidopsismiR156a and miR156c genes,which are the major source of miR156in young seedlings. Real time PCRrevealed that mature miR156in tobacco exists among different organizations (root, stem, leaf, flower),and declines with successive leaf position.2. The fragments of tobacco miR156c precursor gene and the mimicry156vector constructed fromIPS1gene of Arabidopsis by overlapping PCR were introduced into tobacco viaAgrobacterium-mediated transformation.PCR and PPT screening showed that the desired genes wereinto the tobacco genome.3. Results of Stem-loop qRT-PCR showed T1generation of transgenic plants have drasticallyaltered expression of mature miR156.4. Over expression of nta-miR156c and mimicry156in tobacco caused a drastically alteredphenotype, with the morphological variation of plant type, leaf architecture, density and size of stomaand flowers.5. Detection of miR156levels and the later veins number in continuous leaf position of the threegenotypes suggests that they have negative correlation. We speculate that the fluctuant endogenousmiR156levels with age contribute to the timing of appearance of the second veins.6. Qualitative RT-PCR of putative target gene and downstream MADs-BOX genes providesreliable molecular evidence of miR156regulating tobacco growth and development. 7. The influence of anthocyanin and chlorophyll in transgenic plants reveals the function ofmiR156in regulating tobacco physiological and metabolic synthesis.8. In a word, we show that the model of miR156tightly regulate tobacco developmental phasechange, physiological metabolism and veins development.