The Full-length CDNA Cloning And Functional Analysis Of OguCMS-related Genes In Cabbage (Brassica Oleracea Var.capitata)

On the basis of the open reading frames of OguCMS-related genes BoMF1and BoMF3from Brassica oleracea in our laboratory previously, the two genes’ full-length cDNAsequences were cloned by RACE technology. In addition, the regulative sequence of BoMF1promoter was cloned by genomic walking(Tail-PCR). In order to study the promoter function,The plant expression vector pBI121-BoMF1P was transformed into Arabidopsis thaliana andidentified the period and place in the plant. And the antisense and interference plantexpression vectors of BoMF1and the interference plant expression vector of BoMF3weretransformed into Arabidopsis thaliana to verify their specific function. Finally, BoMF1’ssubcellular localization was achieved by using the techniques of vector construction and genegun. The main conclusions of this thesis are as follow:1. The full-length cDNA was cloned by RACE technology according to the open readingframe of BoMF1. The full-length cDNA is626bp, which conains a length of42bp of5’untranslated region,137bp of3’ untranslated region and a477bp open reading frame,encoding148amino acids.2. The regulative sequence of BoMF1promoter from Brassica oleracea was cloned bygenomic walking(Tail-PCR). In silico analysis showed that this sequence contained antherdysregulated element TGTGG and GTGA, and the two key sequence were consistented withA9promoter. In order to study the promoter function, the plant expression vector wastransformed into Arabidopsis thaliana and identified the period and place in the plant. It wassuggested that the BoMF1promoter could drive the GUS gene exclusively express in antherand pollen at the later stage of Arabidopsis thaliana development.3. The antisense and interference expression vectors of BoMF1were transformed intoArabidopsis thaliana and transgenic rate were0.367%and0.465%. the gene was associatedwith anther development presumably by observing the plant phenotype of Arabidopsisthaliana T1generation. The T2plants showed a ratio near15:1of resistance oversensitive,which was in accordance with Mendel’s law of inheritance, presumably there wereabout2transgenes integraged into the genome of Arabidopsis thaliana.4. The subcellular localization of BoMF1was cell nucleus by using the techniques ofvector construction and gene gun.5. The full-length cDNA was cloned by RACE technology according to the open reading frame of BoMF3. The full-length cDNA of is1247bp, inculding a length of37bp of5’untranslated region,178bp of3’ untranslated region and a1032bp open reading frame,encoding343amino acids.6. The interference expression vector of BoMF3was transformed into Arabidopsisthaliana and transgenic rate was0.320%. The genes was associated with plant sterilitypresumably and laid the foundation for further research on its function through theobservation plant phenotype of Arabidopsis thaliana T1generation.