Sequence Analysis And Expression Characterization Of AP1(APETALA1) Gene From Sedirea Japonica

Sedirea japonica is a plant of Orchidaceae Sedirea. With its small,simple and elegant, and wonderful fragrance,long flowering flower,and easy survived,Sedirea japonica is a epiphytic orchid with high ornamental value. In recent years,molecular biology has been widely used in flower related researches and the flowerbreeding process, and these lead the genes of Orchidaceae related especially relatedflowering and their expression to be a hot spot of current research, but the research onSedirea japonica is considerably rare. In this study,the materials is petals of Sedireajaponica, the methods are mainly RT-PCR and RACE technique. The conserved sequenceof AP1（APETALA1） gene which is related flowering is cloned, and the full length of thegene is amplified, and then,find the open reading frame,and analysis the sequence bybio-information; real-time fluorescence quantitative PCR is used to analysis the expressionof the gene in different periods and different parts of the development; and pBI1304-EJAP1as a Sedirea japonica AP1（APETALA1）gene plant expression vector has beenbuilt.The main results are as follows:1Clone the full cDNA sequence of AP1and sequence analysisExtracted total RNA from petals of Sedirea japonica when it was flowering by usingplant total RNA extraction kit.The conserved sequence of AP1（APETALA1） gene wascloned, and the homology to Phalaenopsis, Cymbidium, Oncidium is99%,90%,88%,respectively. The full AP1gene sequence was expanded by5’ and3’ RACE, thelength is1221bp with the ORF of753bp,the5’non-coding regions of66bp, and the3’non-coding regions of402bp. The gene encodes250amino acids. Sedirea japonica AP1gene amino acid physicochemical properties and homology analysis and phylogenetic treewas constructed, conserved regions of secondary structure analysis and prediction bybiological information analysis method the.2Build Sedirea japonica AP1（APETALA1） gene plant expression vectorpCAMBIA1304-EJAP1.Double enzyme digestion the pMD19-EJAP1and pBI221by restriction enzymes SmaⅠ/XbaⅠafter they are sequenced to be right,connected by T4DNA ligase to build the intermediate vector plasmid pBI221-EJAP1. Then,double enzyme digestion thepBI221-EJAP1which enzyme digestion results are right and pCAMBIA1304by restrictionenzymes Pst I/EcoR I. Recycle the purpose gene fragments, and connect them withpCAMBIA1304by T4DNA ligase. Recombinant expression plasmid pCAMBIA1304-EJAP1could be got. After it is verified by colony screening and double enzymedigestion,the plant expression vector pCAMBIA1304-EJAP1of the Sedirea japonica AP1（APETALA1）gene is built. To lay the foundation for genetic transformation and hybridverification.3Analysis spatiotemporal expression of the Sedirea japonica AP1（APETALA1）gene byRT-PCRExtracted RNA from petals of Sedirea japonica in different periods and different parts,then reverse transcribed the RNA into cDNA by real-time PCR. The relative expression ofthe target gene can be figured out according to Rel. Exp=2-ΔΔCtformula. It was found thatSedirea japonica AP1gene can be expressed with the features of different expressionabundance in all the seedling stage, bud and flowering stage.This study found thatexpression levels in scape and buds is higher than roots and leaves in the seeding stag andbud stage, especially in scape. And in the flowering stage, the highest expression part isalso scape. And in this time, expression levels in roots is higher than leaves. The gene isalso but trace expressed petals, sepals and lip, and highest in scapes, stigma ranked second.This study results show that Sedirea japonica AP1APETALA1gene not only plays animportant role start floral meristem and floral organ differentiation,but also participate inthe development of other organizations.