Cloning And Function Analysis Of Dominant Genic Male Sterility Related Genes And Their Promoters In Cabbage (Brassica Oleracea L. Var. Capitata)

Crop of the Cabbage group are worldwide cultivated vegetables. Hybrid seeds are adopted widely in Brassica species for its great contribution on the production enhancement. Male sterility is a very useful alternative strategy in hybrid seeds production in avoiding the labor consuming manual cross-pollination, and it is also advantage on the seed quality assurement. In recent years, although an increasing number of studies have been focused on characterization of microspores and pollen formation from cytological and biochemical aspects, and the description of the abnormal phenomenon in meiosis and microspore development have been reported, knowledge about molecular aspects of pollen abortion is still rather limited. Cloning and characterization of the genes related to male sterility maybe a useful tool in investigating the molecular mechanisms of microsporogenesis.Three different TDFs (Transcript Derived Fragments) from male sterile and fertile lines of cabbage were used to clone their full length cDNA sequences as querying probes. The sense and anti-sense sequences of genes BoRALFL1 and BoPMEI were used to construct the plant expression vectors, and were transfered into Brassica campestris and Arabidopsis plants by Agrobacterium-mediated method. The functions of these genes in pollen formation were investigated. We also cloned the 5'untranscription regions of genes BoRALFL1 and BoPMEI, constructed two plant expression vectors with 5'sequences as promoters, and analyze the promoter activity of these sequences with transient and stability expression systems. Highly efficient expression, purification of recombinant protein BoPMEI and its activity against enzyme PME was performed. Main conclusions are listed as follows:1. The cDNA sequence of gene BoRALFL1 (GenBank accession number DQ059310 ) was 490 bp in length, and contain an 240 bp obvious open reading frame (ORF) , which encodes a 79 amino acids peptide. Homologous analysis shows that the amplified cDNA has 82% identity on the nucleotide acid sequence, and 56% identity on the amino acids sequence with Arabidopsis gene RALFL8. Further analysis shows that the pepetide is possibly a preprotein with a signal pepetide and multi-phosphorylation sites, and the C-terminal amino acids of the pepetide are highly conserved among different plant species.The full-length cDNA of gene BoPMEI (GenBank accession number DQ116449) was...