Cis-acting Elements And Trans-acting Factors Of BnCYP704B1 Gene Related To Pollen Development In Brassica Napus

The BnCYP704B1 gene encode a fatty acid oxidase which specific expression in anther tapetum,and control male fertility trait in the male sterile two-types line S45 AB.To do this research,the promoter truncation experiment and bioinformatics analysis were used, combined with yeast hybrid technology, we analysised transcription factors which related to pollen development.The results of the study are as follows:1 Promoter analysis of the gene BnCYP704B1Amplificate upstream section of BnCYP704B1 gene,we regarded 1085 bp infront of initiation codon as its promoter. Based on the bioinformatics prediction,we devided the sections into 616, 466, 340, 466, 101 and 80 bp(Pms1616, Pms1466, Pms1340, Pms1203,Pms1101, Pms180) by 5’end deleted and fuse the fragments to GUS reporter gene, Using agrobacterium-mediated transformation for above reorganizated carrier and negative contrasts Pck,and GUS histochemical staining to positive transgenic plants.Results show that: GUS gene were driven by deletions of Pms1616, Pms1466, Pms1340, Pms1203,while Pms1101, Pms180 and Pck had no expression of GUS gene. It means that there were several important regulatory elements locate within-202 bp ~-103 bp, controlling BnCYP704B1 gene specific expression in anther.2 Research of yeast one-hybrid about BnCYP704B1 in Brassica napusConstruct of yeast one-hybrid bait vector Pro45-AbAi based on above cis-acting element,we tested the bait strains self-active concentration for AbA expression,determined that the Pro45-AbAi has a minimal inhibitory concentration of 100 ng/mL AbA. Screening rape flower bud cDNA library with 100 ng/mL AbA, there are three complete MYB transcription factors were selected and clone those homologous gene in Brassica napus. Through DNA and protein point to point verification, proved these three genes have interacted with P45-AbAi’s special segment.3 The genetic testing of the possible regulation genesWe focus on the possible upstream regulation genes of BnCYP704B1. Their homologous gene in Arabidopsis thaliana are at4g17695,at3g12730 and at4g13640,respectively. Through homozygous identification by PCR,observing the phenotype of homozygous mutant to detect whether there were any abnormality in the development. we found that all mutants showed fertile phenotype.