| Genic male sterility (GMS) systems are regarded as a promising alternative to CMS, due to the stable and complete male sterility and no negative cytoplasmic effect on yield. As an effective and economical pollination control system, GMS are convenient for the production of hybrid seeds. Nevertheless, the mechanism of male sterility has not been completely discovered. Over recent years, significant progress has been made towards understanding the process of pollen development, and much of this has come from the use of male sterile mutants in model system Arabidopsis. The advent of the genome availability and the development of tools for analysis of gene function mean that such methods can now be carried out in Brassica napus.In the present research, light microscopy and transmission electron microscopy techniques were performed to investigate the defects in genic male sterile plant Rs1046A and 9012A from microsporogenesis stage to male gametogenesis stage.Two differentially expressed ESTs from suppression substractive hybridization (SSH) and cDNA microarray between Rs1046A and B were used to obtain full length cDNA and genomic DNA by 5'/3'RACE technology and Genome Walking. RT-PCR and in situ hybridization were conducted to analyze the spatially and temporally expression pattern of these genes. In addition, antisense suppression vector of BnQRT3 and BnATA20 gene were transformed into Brassica napus. The functions of both genes in anther development were studied in detail. In the meanwhile, we analyzed the cis-elements from the promoters of both genes, and detected the activities of them according to stable expression systems. Main conclusions are listed as follows:1. The main sterile stages and characteristics of two male sterile plants were clarified by cytological observations. Rs1046A (DGMS) occurred from premeiosis stage to meiosis stage. Sporogenous cells did not undergo meiosis and become "cotton-clump" structure denoted by abnormal microspore. Additionally, the degeneration pattern of tapetal cell in sterility appeared very differently from fertility. As for 9012A (RGMS), the abortion initiated at the early tetrad stage. The sterile tapetal cells swelled with expanded vacuoles and finally filled the center of the locules where a majority of tetrads collapsed and degraded.2. On the basis of differential expressed EST:2-C15 (GenBank:EE392320) and 1-H16 (GenBank:EE392282), the cDNA sequence and genomic DNA of BnQRT3 (2-C15) were obtained by 5'RACE and 3'RACE and Genome Walking. The major open reading frame of BnQRT3 transcript was 1428 bp, encoding a 476 amino acid protein. Target P 1.1 predicted BnQRT3 was in a secretory pathway. Based on the comparision with plant PGs that functional assays have been reported, BnQRT3 belongs to which is thought to encode polygalacturonase activity and derived from genes expressed mainlv in flower buds. The 1559bp cDNA and 2907 bp genomic DNA of BnATA20 (1-H16)were isolated by in silico cloning and genome walking. Because the 445 amino acid encoded by BnATA20 contained (GXGX)n glycine-rich repeat, we regarded it as GRP (glycine-rich protein).3. RT-PCR analysis indicated that the BnQRT3 mRNA was found in stamens, ovaries, sepals and petals in both fertile and sterile plants, but not found in leaves or in tender stems. In situ hybridization analysis demonstrated the transcription of BnQRT3 in the anther occurred in the tapetum from the microsporocyte stage to binucleated pollen stage, and also can be detected in microspore mother cells, tetrads masses and mature pollen grains. BnATA20 transcripts could be exclusively detected in the tapetal cells from the tetrad stage to binucleated pollen stage by in situ hybridization. RT-PCR analysis showed that no mRNA was found in the other tissues except the stamens.4. The antisense vector and RNAi vector of BnQRT3 gene were transferred into Arabidopsis Columbia and Brassica napus Huashang 5 individually. Male sterility were observed in T1 progeny of BnQRT3 inhibited plants in both species. BnQRT3 expression was inhibited in these male sterile transgenic plants. Under light microscopy and scanning electron microscopy, there are two different phenotypes of sterility between antisense suppression and RNAi:1. A minority stamens of BnQRT3 RNAi transgenic Brassica napus and Arabidopsis contained numerous shriveled pollen grains with deep invaginations, resulting in the poor pollen vitality. In vivo germination test suggested pollen tubes extension were delayed in the style.2. The stamens from 12 antisense suppression plants and 3 BnQRT3 RNAi plants were completely aborted. According to the observation of transverse-section from sterile anthers, we found BnQRT3 gene, when disrupted, caused premature tapetal degeneration and complete microspore abortion, as well as a reduction in filament elongation.5. The BnATA20 antisense suppression vector was transformed to Brassica napus according to the Agrobacterium-mediated method. In T1 progeny, the sterile plants which do not produce pollen could be detected. In contrast with the fertile stamens, no BnATA20 transcript expressed in the transgenic sterile stamens. In the light of cytological analysis, we could find aberrations in the tapetum when BnATA20 was knocked-out, further leading to microspore cell death. BnATA20 was predicted to specifically involve in the tapetum proliferation.6.992 bp region upstream of the BnQRT3 transcript start sites was further analyzed by searching for putative regulatory motifs using PlantCARE and PLACE. A number of motifs involved in pollen-specific expression. The activity of the BnQRT3 promoter was detected by histochemical staining of GUS activity in BnQRT3Promoter-GUS transgenic Arabidopsis and Brassica napus plants. Strong GUS expression was observed in floral organs including the a series of developing florets from the pollen mother cell stage to anther dehiscence stage, stigma, vascular tissue of filaments, veins in sepals and petals, branch connective and flower abscission zone.1149bp BnATA20 promoter-GUS transgenic plants showed GUS expression exclusively in stamens. The two promoters BnQRT3P and BnATA20P conferred an identical GUS expression pattern on different regions associated with excision and wounding, suggesting that both of them are responsive to wounding. Additionally, we noted the localization of both promoter-GUS expression correlated tightly with the aerial vascular systems including petioles, midribs and stem either by compression with forceps or excision. In the region of BnATA20 promoter, we could find several putative wound-responsive elements specifically expressed in vascular systems including the W-box, W1-box and GC box. However, these elements did not exist in the BnQRT3 promoter. |
PDF Full Text Request