Male Sterile Phenotype Analysis And Molecular Basis Research Of The Transgenic Mutant Tobacco

Tobacco is an important biological model plant; it is the important economic crops in the world, so the study of genetic function of tobacco to the plant biology and agricultural economy has an important significance. In the model plant Arabidopsis, use the male sterility mutants to investigate the molecular mechanism of the progress in pollen grain development has been proved to be a direct and effective strategies. We can get a lot of male sterility mutants at a time through T-DNA insertion method. As long as the material quantity is large enough, it is possible to research the functions of tobacco male sterility genes with this method of creating mutant library. So the research for the existing tobacco male sterility mutants has the vital significance. Thus, the study on gene function of tobacco has an important effect on plant biology an agricultural economy. At present, the sequencing of the genome of the two diploid tobacco species which are considered to be the ancestors of the tetraploid tobacco(Nicotiana tabacum) have been finished. This provided abundant statistics and research platform for the study on gene function of tobacco. In this study, a T0 generation of a transgenic male sterility tobacco mutant with a T-DNA insertion is the research material,and the methods of morphology, cytology and molecular biology was used to research its different with the wide type. We used the Tail-PCR technology separate the T-DNA flanking sequences successfully. Based on the information provided by the T-DNA flanking sequences,we used the forward and reverse genetics approach to research gene function leading to the male sterility. The main results are as follow:(1) Morphological study found that there were no obvious differences between the mutant and wild type tobacco during vegetative growth stage. But in the reproductive growth period, the mature anther of the wild type was green, large and filled with well developed pollen grains. However the anther of male sterile mutant tobacco was dark brown, small and appeared to lack pollen grains, meanwhile most of pollen grains in the anther were shrunken and malformed. The wild-type floral organs were significantly longer than the mutant. The filaments were not shorter than stigma in the wild type but that were shorter than stigma in themale sterility. Statistical results showed that the average number of pollen grains in one anther in mutant was less and most of the pollen grains were shrunken and malformed with a low viability. In vitro culture germination, the percentage of fertility was as low as 10.6%.(2) Used the DAPI staining method to deal with buds and determined the corresponding relation of bud length and pollen development periods in the wild type and mutant respectively. This work laid a certain foundation for later of cytology and molecular biology research. We also found that the length of flowers in mutant were shorter than that in the wild type tobacco in different development period.(3) The observation of cytology combined with DAPI staining results indicated that the dysplasia of the male gamete in male sterility mutant occurred during the mononuclear stage when the tetrads were released. The characteristics were that when microspores were released into the anther locule, part of them still adhered to each other due to the continued presence of callose walls. This part of microspores could not continue to develop and eventually degradation. During the binucleate pollen period, a lot of the pollen grains in mutant occurred vacuolation and could not complete the progress of mitosis, then in the later development, the cytoplasm of these pollen grains depredated constantly until only left a little contents or just a shell when the rest of the few pollens were matured.(4) We surveyed the wall of the mature pollen grains using the scanning electron microscope and found that the wild-type pollen wall were smooth, but the pollen wall of male sterility mutant had a wealth of surface decoration, mostly presented stripe shape and a few dot sag. Then we used the transmission electron microscope to do the further research and found the bacula seemed abnormal, and maybe it caused the following problems such as deposition problem of sporopollenin.(5) Used the mutant as female and male parent respectively to cross fertilize with the wild type. The T1 generations were all fertile and then we guessed that the sterility phenotype was controlled by the nucleus. But we did not find male sterility tobaccos expected after getting the self-cross of T1 generation up to now. Maybe this is because the statistical number too few, we had been expanded the planting quantity to expect get the result.(6) The T-DNA flanking sequences were isolated by thermal asymmetric interlaced(TAIL)-PCR. When we used the left border(LB) and right border(RB) primers, we obtainedone fragment of 500 bp and two fragments of 1.2 kb and 900 bp respectively. Based on the information from NCBI, we found that there were two fragments belonged to one undefined large fragment of the genome of Nicotiana tomentosiformis. Then we chosen three candidate genes to do the later woke. One of them was X transcription factor and the T-DNA insertion induced a lose 60 bp of it?s promoter. The other two were both unknown function genes, one of them could be destroyed in it ? s 3 ? UTR region and another was only known a part of transcribed sequence, the full-length sequence remained to be amplified.(7) We did the quantitative analysis of the three candidate genes by real time fluorescence quantitative PCR in wild type and mutant tobacco?s different organs respectively and found that the gene of X transcription factor had an obvious difference in the two tobaccos. Then we do a further research on the three candidate genes expression in different development period of the anther of the two tobaccos; found that the expression level of X transcription factor gene in the mutant tobacco was significantly lower than that in the wild type either in the organs or in the whole process of anther development. Meanwhile, the relative expression level of unknown gene in mutant was far lower than the wild type during the period of mononuclear pollen grains. So we guess that the sterility could be caused by the reduction expression of X transcription factor gene or unknown gene.