| To increase the yield of crops in a large scale, the potentiality of traditional breeding and cultivation technology is rather limited so far. Therefore, a new agricultural revolution namely genetic modified crop is indispensable to solve the problems of the future food security in China. However, the integration mechanism between foreign gene and the plant genomic DNA is still an enigma about the transgenic plant. Especially few endevours had been done to discover the mechanism of the pollen tube pathway transformation method, which was developed by Chinese scientist. On the basis of the achievement of the Bt cotton, this study, on the one hand, had developed vgbM (modified Vitreoscilla hemoglobin gene) transgenic plants and evaluated the effect of the GM crop on yield improvement. On the other hand, the integration loci of the insecticidal gene on GK12 Bt cotton were analyzed. The result will be helpful to the studies in understanding the transformation mechanism, improving gene transfer efficient, and the site-specific integration by homologous recombination.According to the amino acid sequence and plant preference codon usage, vgbM gene was designed and synthesized and the plant expression vector pGBI4ASVHB was constructed successfully. Studies found that the transgenic tobacco plants obtained by agrobacterium-mediated transformation exhibited enhanced growth and showed more flowering rates. Meanwhile, the bloom date moved up. Furthermore, transgenic plants contained, on average, 23.4% more chlorophyll in leaves. A bivalent plant expression vector pGBRABV carrying both vgbM gene and Bt insecticidal gene was constructed in further. Seven transgenic lines were obtained by pollen tube pathway transformation. The transgenic cotton plants had showed obvious cotton bollworm resistance. Simultaneously, we also found their geminating speeded up, leave chlorophyll content improved by 13%, flowering date moved up and the boll number increased significantly. This work had proved that the transgenic cotton expressing Vitreoscilla hemoglobin showed both yield improvement and water logging resistance. We also noticed that chlorophyll content was a good mark to judge the expression of Vitreoscilla hemoglobin in transgenic plant. This work had laid a research foundation for developing yield improvement transgenic crops in further.This study also developed a genome walking method based on single chain PCR and nested PCR. Utilizing the method, the integration flanking sequence of the insecticidal gene in GK12 cotton was analyzed. We found the CrylA expression box had recombined with other foreign DNA fragment. The result had supported the 'Two phase integration mode'. Meanwhile, we found some evidence for that topoishomerase and transposon may involve with foreign DNA integration process. We also proved foreign gene harbored in a normal small plasmid can be transformed successfully by pollen tube pathway transformation. This suggested that a vector-free transformation is feasible. So it is beneficial for GMO safety evaluation. More important, it is favorable for site-specific integration (gene targeting) through homologous recombination without the vector impediment. According the integration flanking sequence and considering the know factors that may influence foreign gene expression, if the foreign gene can be targeted on a 'stable-expression' position directly, the tedious screening procedures during GM crop development could be simplified. The setup of a vector-free new plant transformation mode will undoubtedly promote the studies and the commercialization of the GM crops.
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