Cloning Ang Analysis Of Differential Expression EST In The Process Of Forming Adventitious Roots Of Tr1pterygium Wilfordii

Tripterygium wilfordii Hook.f is a kind of plant with insecticidal activity and medicinalvalue. It has toxic action, antifeedant and development inhibition to many pests; in themedicine study, it has many activities, such as antitumous, antibacterial, anti-inflammatory.With the dramatically increase demands in medicine and biorational pesticide, wildT. wilfordii has been exploited blindly, and so, it brings a lot pressures to the environment.Adventitious root is the root which is generated in tender stem, branch and so on, and thesource is not fixed. Compared with conventional artificial cultivation of root, adventitiousroots have many advantages, such as: fast-growing, free of season, and high cotent ofsecondary metabolites. So, in recent years, people have paid more attention to producesecondary metabolites with adventitious roots culture. In this study, to explore the formationmechanism of adventitious roots of T. wilfordii, we use leaves and callus to induceadventitious roots, and then, we clone and analyze differentially expressed fragments in theprocess of adventitious roots formation of T. wilfordii, with molecular markers technology ofcDNA-SRAP, and then, we predict their functions. The main results are as follows:1. Regarding leaves and callus of T. wilfordii as explants, the adventitious roots areinduced. Then, through optimizing the culture medium and auxin levels, we have screened thebest medium and auxin to adventitious root of T. wilfordii, which is MS+1.0mg/LIBA.2. After transferring the well-grown callus to MS+1.0mg/L IBA rooting medium, thetotal extracted RNA of0d,2d,4d,6d,8d,10d,12d are obtained, and then, we reverse-transcribe them into cDNA. After that, using orthogonal design, we have optimized the bestcDNA-SRAP reaction system which fits to the formation of adventitious root, the volume is25μL:1.0UTaq enzyme,0.2mmol/L dNTPs,2.0mmol/LMg+,40ng cDNA, primers0.4μmol/L each. Using the optimized reaction system, we have screened13pairs primers, which arepolymorphic, clear, high repeatability. And then we use the13pairs primer to analysis thecDNA-SRAP, finally,36different sequences have been obtained by sequencing.3. Through the blast comparative analysis to36sequences, we have received6fragmentsrelated with the formation of adventitious root of T. wilfordii. Among6fragments, the protein of YS37-1has a high similarity with mitogen-activated protein kinase(MAPK), and itprobably participates in the approach to the forming of adventitious root of T. wilfordii, underthe influence of auxin; the homologous protein of YS44-1is a kind of IAA amino acidhydrolase, and the hydrolase of YS44-1is probably related with the formation of adventitiousroot; the homologous protein of YS34-1is a kind of Homeobox protein leucine zipper, and inthe experiment of inducing the adventitious root of T. wilfordii with IBA, it is probablyinvolved in the signal transduction pathway of auxin, indirectly influencing the formation ofadventitious root.