Screening And Cloning Of Genes Involved In Bulbil Development From Pinellia Ternata Thunb.Breit

In this thesis,transcriptome and profiles sequencing of Pinellia ternata Thunb.Breit were completed.The members of three subfamily of PEBP family(PteFT,PteMFT,PteTFL1)were identified on the basis of sequencing.The RACE method was used to full-length cloning and bioinformatics analysis of the PEBP family members.The relative expression levels for these three genes in different developmental stages of bulbil were analyzed using qRT-PCR.This was provided a reference for study the PEBP family genes in P.ternata and provided a theoretical basis for the molecular mechanism of bulbil development.Results of these studies are listed as follows:1.The different expression genes between petiole,bulbil primordium and expanding bulbil were screened on the basis of profiles sequencing.As a result,4785 differential unigenes were obtained,and 3702 unigenes were annotated in KEGG,GO,or NR database.There were each 2 unigenes in the MFT-like and TFL1-like subfamily.Furthermore,9 unigenes of PEBP family were annotated in transcriptomic data: 6 unigenes belong to the FT-like subfamily,2 unigenes belong to the MFT-like subfamily,1 unigene belong to TFL1-like.2.The RACE method was used to gain full-length cDNA sequences.Finally,the full-length sequences of PteFT,PteMFT and PteTFL1 genes were 759 bp,969bp and 1152 bp,respectively.Three genes encode 174,207 and 174 amino acids,respectively.Alignment with homologus sequences from other species,showed that CeFT(Cymbidum ensitolium)was the highest homology with PteFT gene,VvMFT(Vitis Vinifera)was the highest homology with PteMFT gene;TgTFL1(Tulipa gesnerlana)was the highest homology with PteTFL1 gene.3.Results of bioinformatics analysis for the three PEBP family genes showed that physicochemical properties,signal peptide,hydrophilicity,secondary structure,homology analysis and phylogenetic tress of PteFT gene were closer to CeFT(CeFT promote plant flowering).It was speculated that the function of PteFT gene was more likely to promoting flowering of plants.Using the same method to analyze the PteMFT and PteTFL1.The results show that PteMFT and PteTFL1 were significantly differed from its homologus sequence respectively.It was speculated that the function of PteMFT gene was different from the GmMFT gene(participate in seed germination)of soybean.It was speculated that the function of PteTFL1 gene was different from the TgTFL1 gene(inhibiting plant flowering)of Tulip.4.The results of qRT-PCR analysis showed that the expression level of PteMFT were significantly different between petiole and bulbils of different development stages,and there were also significant differences between bulbil materials in four developmental stages.The relative expression of PteMFT gene was increasing first and then decreasing.Combining the expression analysis results of MFT-like gene with other species,it was suggested that the PteMFT gene may be related to bulbil primordia inducing and bulbil growth;Quantitatives of PteTFL1 gene expression had significant differences between petiole and bulbils primordium formation period,bulbils primordium development stages,bulbil expansion stage.Furthermore,expression of PteTFL1 gene was increasing first and then decreasing from bulbil primodia formation to bulbil ripeness.Combining the expression analysis results of TFL1-like gene with other species.It was speculated that the PteTFL1 gene may only related to the early bulbil primodia formation.However,the PteFT gene only was expressed in the petiole and rarely expressed in any bulbil materials from different developmental stages.It was speculated that the PteFT gene didn't participated in bulbil development and may involved in flowering regulation acted as other FT-like gene.