| Oilseed rape is an important oil crops in China and all over the world. Oil rape is of obvious heterosis for biomass and seed yield. Pollination control systems comprising of different fertility control genes are at present main technology for utilization of heterosis in crops. However, there are just a few of functional genes cloned and identified, and understanding of molecular mechanisms of male gametogenesis is quite poor. The present study identified functions of three fertility-related genes using efficient amiRNA (artificial microRNA) interference technology and investigated certain molecular mechanisms of male sterility caused by the genes. These studies open a window for gametogenesis regulation and construction of new pollination systems.Technical route of the study is: The gene profile of heterozygous dominant digenic male sterility lines on microarray of the Arabidopsis whole genomes was used to select candidate genes which was differentially expressed in the lines. The selected genes were investigated by qPCR for their expression patterns in different organs of oilseed rape plants. The genes with specific expression in anthers were studied for their functions by amiRNA interference technology in which sequences of amiRNA interference vectors harboring herbicide-resistant gene bar were designed based on their homologous counterpart in Arabidopsis. The target sequences in these vectors were transformed into Arabidopsis by flower dipping methods. Consequently, seeds from transformed flowers were germinated and screened out for the herbicide resistance, and observed for phenotypes. Finally transgenic plants with male sterility were probed into molecular mechanisms behind.The major research results are as follows:1. Establishment and application of artificial micro-RNA interference technology systemTwo target mRNA sequences were selected and constructed into microRNA vectors by using the amiRNA silencing technology. Arabidopsis were transformed with these vectors by flora dip method and screened out by herbicide. All of the 8 transgenic Arabidopsis showed microspore aborting, mature pollen shrunken and reproductive ability was lost, which indicated the male sterility or semi-fertile phenotypes. A very similar result was obtained by our research and the published paper of Etienne 2009: both of the amiRNA technology and T-DNA inserted mutants and RNAi transgene resulted in the same male sterility phenotype. This indicated that amicroRNA technology as a means of studying gene functions is feasible and reliable. Artificial micro-RNA interference technology system and identification system was established by research work above which providing an effective technological strategies for studying rape microspore development-related functional genes.2. Clinging and transgenic analysis of a new microspore development regulator: PDF1PDF1 is a gene with unknown function. Microarray results showed that the expression level of BnPDF1 was very different between fertile and sterile plants. In this study, quantitative PCR was used to verify the expression profile of BnPDF1 in roots, stems, leaves, flowers, siliques, pistils and stamens, showing that it highly expressed in the stamens of fertile plant. Expression level of BnPDF1 in stamens and anthers of oilseed rape was correlated to male fertility significantly; PDF1 is involved in male gemetogenesis. So, PDF1 homologous genes in three phylogenetic species, which were Brassica napus, Brassica rapa and Brassica Oleracea were cloned and a variety of bioinformatics analysis and homology comparison were performed. The result of alignment and structure prediction shows that these three homologous genes have a similar function with AtPDF1. After that, a subcellular localization AtPDF1: GFP vector was constructed and bombarded into the onion epidermal cells which confirmed that PDF1 located in the cytoplasm membrane.On the above basis, we constructed amiRNA interference vector which was then transformed into Arabidopsis by the flower dipping method. After screening with herbicide and PCR detection, 97 transgenic plants were obtained. T1 transgenic plants phenotype showed that 61 plants displayed complete or partial male sterility: shrinking anthers with no pollen or with dramatically reduced microspores and significantly reduced number of pods. Northern blot showed that the interfering sequence 21bp for PDF1 was expressed in transgenic Arabidopsis plants. Quantitative PCR PDF1expression levels were not detectable or dramatically decreased in transgenic Arabidopsis plants, caused by the 21bp interfering sequence. Based on the above results, interference of AtPDF1 gene led to male sterility. PDF1 is a new functional gene which can regulate microspore development and anther fertility.3. PDF1: a novel PCD inhibitor plays a crucial role in and plant growth and development.To further investigate molecular mechanism of PDF1 controls anther development, transgenic Arabidopsis was analyzed histologically, ultrastructurally and molecularly. Cytological observation on the transgenic plants indicated that abortion of microspore development occurred at the stages of from tetrads to uninucleate. During this period, the nucleus of microspore abnormally degraded, and afterward no nucleus were observed, suggesting occurrence of PCD. DNA ladder test confirmed that genomic DNA had been segmentalized or degraded, one of PCD characteristics. In addition, transgenic plants displayed significant dwarf phenotype. Accordingly we observed the stems and leaves of transgenic plants by fluorescence staining, and found out the outer mesophyll cells and epidermal stem cells also appeared the phenomenon of nuclear degradation. Q-PCR analysis showed that expression levels of PCD inhibitor-like genes such as MS1, FBR and SPT were decreased remarkably while PCD promoter -like genes such as MMD and DAD were increased in transgenic Arabidopsis in which expression of PDF1 were almost totally inhibited.From these results we inferred that PDF1 is a new PCD inhibitors, which inhibits PCD initiation in normal cells to ensure the cell differentiation, division and normal tissue development. Inhibiting of this gene induces rathe PCD and as the consequence, normal functions or development of cells or tissues are interrupted, one of resultant phenotypes are male sterility and dwarf. Valuable clues for further study on PDF1 function and mechanism was provided by these researches. Further characterization of PDF1 can contribute to the understanding of the molecular mechanism of anther development and male sterility.4. Rape ROP1homologous gene silencing leads to male sterility in ArabidopsisBased on the analysis of Microarray expression on Brassica napus homozygous pairs of dominant male sterile line, a differentially expressed Brasica napus gene (BnROP1) was selected which is homologous with AT3G51300 (ATROP1). Fluorescent quantitative PCR was used to analyze the expression pattern of BnROP1 in different tissues of Brassica napus, which included roots, stems, leaves, flower, pistil, stamen and silicle. The results confirmed that BnROP1was highly expressed in fertile flower and stamen. On this basis, microRNA interference vector was constructed and then transformed into Arabidopsis thaliana by Agro bacterium, obtaining 12 transgenic seedlings in the result. The phenotype of T1 transgenic generation was observed during florescence, most of transgenic seedlings showed that reduction or disappearance of silicles, no pollen, short filaments, deteriorated anther and aborted microspore. This phenomenon affirmatively indicated that these seedlings were fully-sterile or semi-sterile. Q-PCR experiments on many T2 generations of transgenic lines showed that the expression level of ROP1 gene decreased remarkably, indicating significant effect of the interference of the target gene. The correlation of transgenic male sterile phenotype and the silencing of ROP1 expressing represented that ROP1 is not only promote tip growth and maintain growth polarity in pollen tubes but also essential for microspore development.
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