Molecular Mechanism Of Monogenic Dominant GMS In Brassica Napus L.

A novel genic male sterile (GMS) line in Brassica napus L., which was identified by Hybrid Rapeseeed Research Center of Shaanxi Province in 1999, was found to be controlled by a monogenic dominant gene which we have designated as MDGMS. The F1 fertility from any fertile lines crossed with MDGMS segregated and the ratio was close to 1:1. Its fertility, floral organ character were investigated. Course of microsporegenesis and the difference of protein expressing in bud were compared and analyzed between MDGMS and its allelic fertile. RAPD marker linked to Ms gene in MDGMS was found using BSA and has been converted into sequence characterized amplified region (SCAR) marker to aid identification of male-fertility genotypes in segregating progenies of MDGMS in marker-assisted selection (MAS) breeding programs. After RAPD marker sequenced and blast, a homologous gene importinαwas found and its complete genomic DNA and cDNA of ORF was screened out from Brassica napus L. The RNAi vector and antisense RNA vector of importinαgene of rapeseed were constructed and were transformed into rapeseed. The primary results are as follow:1. The MDGMS flowers were characterized by reduced anthers with little withered or no pollen grains and no seed set was observed on selfing. The most prominent change in the male-sterile flowers was a distinct reduction in the length of the stamen filament. However, the gynoecium and nectary did not show any differences in sterile lines compared with that of allelic fertile ones. It can normally set seeds when the male-sterile pistil was pollinated with other fertile pollen, suggesting that the gynoecium of male-sterile was fertile.Expression of male sterility/fertility in two clones derived from a single plant was generally similar in field and greenhouse. These observations based on 48 cloned genotypes suggested that this GMS sterility was very likely controlled by a single dominant nuclear gene without environment effect.2. The microspore development course was observed by paraffin slices, autolysis of tapetal layer took place around the tetrads stage, therefore the nutrition was supplied for the development of the microspore. While in the MDGMS microspores abortion was found before the pollen mother cells meiosis stage, and the tetrads couldn't be formed, then the contents of microspore leaked out, microspore lysis followed after, at last, sterility occurred. These results indicated that the MDGMS male sterility was caused by its development rather than autolysis of tapetal layer.3. The differences of protein expressing in buds between MDGMS and its allelic fertile line was detected at the same stage. The results showed that the quantity of detected protein dots in fertile line is more than that in MDGMS. The molecular weight of special proteins in fertile line is mainly below 60kD, especially below 30kD. While the ones in MDGMS are relative uniform. What the same between MDGMS and allelic fertile line is that the isoelectronic points (pI) of respectively special proteins are mainly in 6.0-7.5.4. Bulked segregation analysis (BSA) was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the Ms gene in MDGMS. Only one RAPD marker OPU031500 was found to closely link to the MDGMS locus in rapeseed. This RAPD marker OPU-031500 was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers SCP1/SCP2 and amplified only one specific 2.3kb DNA fragment in each bulk. There are some mutant loci between the two DNA fragments after sequencing. Both sequences are highly homologous to Arabidopsis Importinαgene. We designed another pair of specific primers SCP3 /SCP4 according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers SCP3 and SCP4. Therefore the RAPD marker was converted into SCAR marker.5. The genomic DNA and cDNA of Importinαgene were cloned by PCR in MDGMS and allelic fertile line in Brassica napus L., including initiation codon ATG and termination codon TGA. The genomic DNA is 2620bp and 2614bp respectively, and cDNA is 1629bp and 1620bp respectively. There are some mutant loci in genomic DNA between MDGMS and allelic fertile line, but only two mutant loci in cDNA. Comparing on the genomic DNA and cDNA, Importinαgene of rapeseed (BIMP a) includes 10 exons and 9 introns.6. Comparing on the induced protein sequence by cDNA sequence between MDGMS and allelic fertile line, Importinαof rapeseed is composed of 542 amino acids, but 540 amino acids in MDGMS. Andαhelix andβturns of Importinαsecondary structure are less in MDGMS than that in allelic fertile line because of two QQ amino acid absence in MDGMS.Protein structure analysis (DNAstar) showed that rapeseed Importinαis consist of a flexible N-terminal Importin-β-binding (IBB) domain and a highly structured domain composed of eight armadillo (ARM) repeats and two HEAT repeats. ARM repeats are the Importinαclassical structural characters and HEAT repeats are Importinβclassical structural characters. So we designated it as BIMP a (Brassica napus Importinα, BIMP a).7. Rapeseed Importinαgene regulates not only oogamete development but also androgamete development. Gene knocking-down plants by RNAi and antisense RNA can't set seeds on either selfing or crossing, indicating that both oogamete and androgamete are sterile. And the gene knocking-down plants are susceptible to downy mildew and virus diseases in comparison to wild type.