| Cre/loxP is a site-specific recombination system from phage PI. Presently, it has been extensively used in the identification of gene function, genome modification, gene expression and gene regulation in plants, animals and microorganisms. Male sterility is an important character in the utilization of hybrid heterosis, and an important aim of plant breeding as well. This work was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male apparatus under the control of Cre/loxP system.1. In this study, we cloned bamase gene from Bacillus amyloliquefaciens. We overcame the strong cell toxicity of barnase gene and successfully constructed a series of plasmids suitable for biolistic and Agrobacterium tumefacien mediated transformation respectively. Positive transgenic plants were obtained by molecular identification.2. According to the results of Paddon CJ et al, we designed two primers of barstar, an strong inhibitor of barnase, and acquired correct barstar gene by PCR, which was then inserted into the binary vector with a view to recover fertility of transgenic wheat and tobacco. Plants transformed with barstar gene were also obtained.3. osg6B, a promoter from rice, can direct gene expressing in anther tapetal during the early developmental stage of microspore from tetrad to uninucleate pollen, with high temporal and spatial specificity. Combining the osg6B promoter and Cre/loxP system, we constructed a set of plasmids suitable for biolistic transformation. By now, selfed seeds from transgenic wheat were obtained and sown in the soil. In addtion, seeds from hybrid crossing between male parent wheat integrated with Cre gene and female parent wheat with blocking fragment were also obtained and sown in the soil. Their fertilities are now been observing and testing.4. In order to control fertility of dicotyledons, we also constructed a set of plasmids containing Cre/loxP system driven by the osg6B and transformed tobacco via the mediation of Agrobacterium tumefacien. Both selfed seeds and hybrid seeds were obtained from male parent tobacco integrated with Cre gene and female parent tobacco with blocking fragment.5. We observed that wheat transformed with sterile gene grown normally, but the amount of pollen decreased significantly after flowering. Consequently, it was hard to produce normal selfed seeds. Pollen vigor testing showed that most of the pollen oftransgenic wheat were sterile. In vitro germination rates of pollen obtained from transgenicwheat were same result, indicating that barnase gene under the control of osg6B can results in male sterility.6. The tobacco integrated with Cre gene was transformed for the second time with binary vector harboring blocking fragment via Agrobactehum tumefacien. Although the vegetative growth of transgenic tobacco followed second transformation was normal, the reproductive organ showed many metamorphosis such as petal spliting abnormally or without spliting, filaments shortening or adhering together, anther spliting abnormally or wilting ealier than normal time. These phenomena were probably caused by the specificity of the promoter or by the leaky expression of bamase gene.7. The staining of pollen harvested from the secondly transformed lines showed different fertility from whole sterility, partial sterility to most fertility, and similar results were obtained from in vitro pollen germination experiments. This demonstrated that, using Cre/loxP system, sterile lines can be obtained through selecting transgenic plants.8. In cloning of barnase gene, we obtained 9 mutants. By analysing these sequences, we deduced that the 14th-aa and 80th-aa of barnase are important for its activity. Meanwhile, we found that the 316th-base is susceptible to mutation and sometimes inserted by an additional C, resulting in the change of the stop codon. These phenomena may provide useful imformation for furture study on barnase gene.
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