Research Of Gene Expression On The Genic Male Sterility In Brassica Napus

Many of genes have their special expression models during plant development. In different tissue or developmental stages under different environments, genes should express at different level. In this paper, we studied the recessive genie male sterile line of Brassica napus by using differential display method cDNA-AFLP to find some genes which were related to male sterility and then provide theoretic basis for the complex regulation mechanism of male sterility. Meanwhile, according to the fertility related genes reported in Arabidopsis thaliana, the primers were to amplify homologous regions in Brassica napus, and the differences between male sterile and fertile plants were compared. The main results were as followed:1. The cDNA-AFLP techniques were optimized and applied successfully to study the mechanism of male sterility. Results manifested that cDNA-AFLP technique, with stabilization and reproducibility, could display the differences between male sterile and fertile plants.2. Three kinds of gene expression models between male sterile and fertile plants were detected. With three-group primers including 512 primer pairs (2*16 ), different numbers bands were generated by each primer pairs, averagely 40 clear bands and maximally 70 clear bands. The polymorphic bands between the male sterile and fertile plants could be divided into three classes, the bands only in male sterility, those only in male fertility, and those in both but with different quantity. The rich polymorphism of bands was helpful to understand the mechanism of male sterility.3. There were 22 cDNA polymorphic fragments which ranged from 100 base pairs to 600 base pairs. After screening, fourteen cDNA fragments were candidates, among which eight fragments originated from male sterile and six from fertile plants.4. Two positive cDNA fragments, 1A-3 and 3B-2 were obtained by analysis of reverse Northern blot and Northern blot, and they were cloned and sequenced. The 1A-3 was highly homologous to a DNA-binding motif of Doublesex- and mab-3-related TF5 from Rattus norvegicus. We presumpted that the gene with the identical DNA-binding motif in plant should be related to pollen development as the DNA-binding motif was very important to sperm genesis of animal. The cDNA fragment, 3B-2, which only had positive Northern blot signal in male fertility, were homologous to an unknown expressed gene of Arabidopsis thaliana. It was uncertain whether its function was concerned with male sterility in Brassica napus.6. Two genes, MS1Bnap and MS2Bnap, homologous to MS1 and MS2 genes of Arabidopsis thaliana were cloned via the method of homologous sequencing. The full length of MS2Bnap gene was 2650 base pairs including seven introns and the length of mRNA was 1928 base pairs. At the present time, only 5' terminus of MS1Bnap covering1755 base pairs with two introns was obtained.7. Sequence analysis indicated single nucleotide differences at five positions existed on MS2Bnap gene of male sterile plants, including two missense and three nonsence mutations. Only one base was different in the first intron in MS1Bnap gene between male sterile and fertile plants. The contributions of those mutations in the two genes of male sterility plant to genie male sterility should be elucidated in further research.