Phenotypic And Molecular Characterization Of Arabidopsis Plants Transformed With A Chimeric Rapeseed Gene BnaA7.HSP70-1-like

The fertility conversion of a genic male-sterile line 9012 AB from rapeseed(Brassica napus L) is controlled by the Bn Ms3/Bnms3 locus and the multiallelic Bn Rf locus which includes three alleles, i.e., the restoration allele Bn Rfa, the male-sterile allele Bn Rfb and the maintainer allele Bn Rfc. Our previous studies have mapped Bn Rfa/Bn Rfb to a physical region of 41-kb on a bacterial artificial chromosome clone, from which a predicted chimeric gene Bna A7.HSP70-1-like has been regarded to be a good candidate of Bn Rfb. In order to validate this speculation and primarily discover the possible molecular mechanism underlying this gene, we transofrmed Bna A7.HSP70-1-like into the model plant Arabidopsis(wild-type ecotype Columbia, WT). The main results are described as following:1. We introduced the expression construct Bna A7.HSP70-1-like-p FGC5941 into WT Arabidopsis plants according to the Agrobacterium-mediated transformation and obtained 25 transgene-positive T1 plants, all of which showed stale and complete male-sterile phenotype. Analysis of the T2 plants generated by crossing the T1 plants with WT Arabidopsis plants showed that the male-sterile phenotype was co-segregated with the transgenic event, indicating that the ecotypic expression of Bna A7.HSP70-1-like can cause the male sterility in Arabidopsis. Further RT-PCR analysis showed that Bna A7.HSP70-1-like was specifically expressed in flower buds of transgenic Arabidopsis plants.2. Results from cytological observations and biochemical investigations showed that there was no obvious difference between the transgene-positive and WT plants during the early meiosis stages. However, the tapetal cells from male-sterile plants became abnormally enlarged and vacuolated at the tetrad stage, and the abnormal anther accumulated less lipid compounds than those from the WT plants. At the young microspore stage of the WT plants, the tapetal cells from male-sterile plants could not differentiate into the secreted type possiblely due to the delayed tapetal PCD process, so they failed to release callase to degrade the collase surrounding the tetrads. Then, the unsepatated tetrads were continuously crushed by the expanded tapetal cells to be degenerated. These results suggested that the Bna A7.HSP70-1-like may interfere the normal differentiation of tapetal cells from around the tetrad stage by delaying the tapetum PCD process.3. We contrastly measured the dynamic anther ROS levels in both Bna A7.HSP70-1-like transgenic and WT anthers through NBT staining of superoxide anion and 2’,7’-dichlorofluorescin(DCFH) staining. The results showed that the ROS level from WT plants gradually increased from stages 6, reached the highest level at stage 8 to 9, and then decreased in the following stages. Similar dynamic ROS level pattern was also observed from the transgenic male-sterile plants; however, the ROS level at each corresponding stage was significantly lower than that of the WT plants. Therefore, the ecotopic expression of Bna A7.HSP70-1-like also has a negative effect on the ROS levels in anthers during stage 6 to 11. In combination with the relationship between ROS level and tapetum PCD, we hypothesized that the reduced ROS levels may be the driving force for the delayed tapetum PCD in transgenic Arabidopsis plants.4. Results from q PCR analyses showed that three transcription factors during late stages for tapetum development like ABORTED MICROSPORES(AMS), MYB80 and MALE STERILITY1(MS1), two putative executors(CEP1 and UNDEAD) for tapetum PCD and one ROS producer(RESPIRATORY-BURST OXIDASE HOMOLOG E, RBOHE), were all greatly down-regulated in male-sterile plants. These results showed that Bna A7.HSP70-1-like may specifically decide the tapetum fate by regulating the downstream genes involved in tapetum differentiation from multiple approaches.5. Through yeast two-hybrid analysis, we found that there is no direct interaction between Bna A7.HSP70-1-like and Bna C9.TIC40-1(Bn Ms3) from the B. napus line 9012 B, 9012 A and T45. These results suggested that Bn Rf and Bn Ms3 may control the fertility conversion in 9012 AB by two independent pathways.In a word, further phenotypic and molecular characterization of the transgene-positive Arabidopsis plants indicated that the ecotypic expression of Bna A7.HSP70-1-like can result in the abnormal development of anther which finally showed a completely male-sterile phenotype, indicating that Bna A7.HSP70-1-like is most likely the target gene of Bn Rfb.