Cytological And Molecular Mechanism Research On The Abortion Of A Thermo-sensitive Genic Male Sterile Line TE5A In Brassica Napus

The photo-thermo sensitive male sterile lines would be widely used in hybrid seed production of Brassica napus for its special advantages. It was very important to study the mechanisms of anther abortion of the photo-thermo sensitive male sterile lines. In this research, cytological biology techniques were utilized to demonstrate the vital abortion periods and cytologicical characteristics of the anther development in a thermo-sensitive genic male sterile line TE5 A in Brassica napus. Then we observed the chromosomal behavior during the meiosis progression by DAPI staining. We constructed a yeast two hybrid cDNA library of flower bud of the thermo-sensitive genic male sterile line TE5 A in Brassica napus by SMART, and screened the host proteins interacting with the BnaA.tsMs protein of it by yeast two-hybrid system. The main results are as follows:1. The filaments of the sterile flower of the thermo-sensitive genic male sterile line TE5 A in Brassica napus were significantly shorter than the fertile’s and the anthers were withered and atrophy. The abortion of the sterile line TE5 A occurred at the meiosis stage. The pollen mother cells could not pass the meiosis, with no dyads and tetrads formed in the sterile line TE5 A. The callose was normally deposited around the pollen mother cells, however, the callose hadn’t been degraded until the mature pollen stage. The tapetum cells were obserbed to be normal in the sterile line. But "pseudo microspores" were formed by the pollen mother cells. The pollen exines of the "pseudo microspores" were abnormal and couldn’t combine with sporopollenin, liposomes and other substances. The "pseudo microspores" were decayed gradually and only empty shells remained at last. The cytological characteristics of the sterile line TE5 A were different from the previous materials. So these indicated that the TE5 A was a novel thermo-sensitive genic male sterile line.2. We observed the chromosomal behavior during the meiosis progression in the thermo-sensitive genic male sterile line TE5 A in Brassica napus. We found that: the chromatins failed to synapse at meiotic zygotene in the sterile line TE5 A and the homologous chromosomes couldn’t start to pair with their homologues. So the synaptonemal complex and the chiasmata(cross-overs) were not formed. The chromatins were not shortened and thickened during the progression of meiosis. Eventually the chromatins were highly concentrated in the central of the nucleus and were decayed at the microspore stage.3. A successful yeast two hybrid cDNA library of flower bud of the thermo-sensitive genic male sterile line TE5 A in Brassica napus was constructed in this study. The capacity of the cDNA library was about 2×107 cfu. The average cDNA inserts were more than 1.8 kb, with a recombination frequency of 100%. The yeast two hybrid cDNA library met the high quality standards of cDNA library, and could be screened by yeast two hybrid system in the following study.4. In this study, we screened the host proteins interacting with the BnaA.tsMs protein of the sterile line TE5 A by yeast two-hybrid system. Forty-nine interactive factors were obtained, which were divided into nine types based on their functions by Gene Ontology. Including: structural constituent of ribosome, DNA binding, protein binding, zinc ion binding, ATP binding, GTP binding, transferase activity, sequenced-specific DNA binding and nucleic acid binding. Three interactive proteins(BUBR1, cyclin-D3, FBW2) of them were associated with meiosis. The three interactive proteins(BUBR1, cyclin-D3, FBW2) might be interacted with the Bna A.tsMs. The fertility of the male sterile line TE5 A could be regulated by the interaction of BnaA.tsMs with the interactive proteins.