The Study Of Cloning, Expression Analysis And Genetic Transformation System Of OnAP1-like Gene In Oncidium ’Gower Ramsey’

Oncidium is part of Orchidaceae plant and representing the hole Oncidium, also knownas Wu Nü Lan or Tiao Wu Lan, originating in the South America and south of North America. Withgood inflorescence branch, beautiful flower shape and bright color, Oncidium has good ornamentalvalue. In recent years, molecular biology has been widely used in flower related researches and theflower breeding experiment, and these lead the flowering related genes of ornamental plant and theirfunction of these genes to be a hot spot of current research, but the research on Oncidium is considerablyrare. In this study, the scape of Oncidium ‘Gower Ramsey’ was used as the experiment material andRT-PCR and RACE technology as technical support. The conserved sequence and the full length ofOnAP1(APETALA1)-like gene which is related flowering is cloned. Then, the open reading frame wasfound, and the sequence analyzed by bio-information. Real-time fluorescence quantitative PCR is usedto analysis the expression of the gene in different periods and different organs. The plant expressionvector of OnAP1-like gene was built and transformed the callus of Oncidium. The main results are asfollows:1Clone the full cDNA sequence of OnAP1-like and sequence analysisA full-length958bp cDNA of AP1(APETALA1)-like was obtained by RT-PCR and RACEamplification the total RNA extracted from the scapes of Oncidium ‘Gower Ramsey’(Orchidaceae) atflowering stage. The gene consisted of690bp gene encoding region which encoding a protein of229amino acids, was designated as OnAP1-like (accession number: KC426946). The analysis of secondarystructure of protein showed that the protein was hydrophilic, and it contained48.91%of α-helix,10.92%of β-sheet and40.17%of random coil. The analysis of protein sequence alignment and clustershowed that the OnAP1-like was close to AP1-like of Cymbidium faberi with an overall sequencesimilarity. The3D structure of the protein expressed by the OnAP1-like gene was predicted.2The plant expression vector pRI101-OnAP1was built.Double digest the plasmid pMD19-OnAP1which sequenced and plasmid pRI101-ON byrestriction enzymes Xba I/Sma I. Connect the product of digestion use T4DNA ligase to construct theplant expression vector pRI101-OnAP1. After colony screening, PCR detection and double enzymedigestion, ultimately determine the plant expression vector pRI101-OnAP1of Oncidium OnAP1-like genes has been built successfully. This can be helpful for genetic transformation of the nextexperiments.3Analysis spatiotemporal expression of the OnAP1-like gene of Oncidium by RT-PCRUsing RT-qPCR to analysis the expression quantity of OnAP1-like of different organs fromOncidium in different stages, the results showed that in different development stages, the expressionlevel of leaves was highest in flower budding stage, while those of root and scapes gradually increasedwith development and obtained a highest expression level at post-flowering stage. In different organs,the expression level of roots was higher than that of leaves before reproductive growth stage; Theexpression level of petals was highest in flower budding stage, and those of scapes was highest inflowering and post-flowering stage. In summary, OnAP1-like gene does play a significant role in thedevelopment and formation of flowers in Oncidium Gower Ramsey.4Genetic transformation of Oncidium ‘Gower Ramsey’ callusBased on the previous researches, the conditions of the agrobacterium mediated transformation ofOncidium ‘Gower Ramsey’ were explored in this study, and a genetic transformation system ofOncidium ‘Gower Ramsey’ was finally identified successfully.