| Crop genetic engineering relies on the introduction of foreign DNA into plant genomes. Although genetically engineered traits provide valuable alternatives to those available through conventional breeding, there is public concern about the consumption of foods derived from transgenic plants. The selectable marker genes are required to ensure the efficient plant genetic transformation, 70 percent of which are genes conferring kanamycin resistance. The presence of antibiotic or herbicide resistant selective marker genes in transgenic plants causes the concerns of the potential hazards on ecological environment and human health. At the same time, the limited number of selectable marker gene prevents the retransformation for genetic engineering. Basically, there are two strategies available for generating marker-free transgenic plants, that is, marker gene avoidance or marker gene excision. The first approach is carry out plant transformation using safer markers or without markers at all. The second approach is marker gene elimination after selections. Among the marker gene elimination strategies, inducible auto-excision presents the most promising choice. Cotton bollworm (Helicoverpa armigera Hubner) is severe insect pest, mainly destroying tomatoes. Insecticidal crystal protein from Bacillus thuringiensis is the most widely-used endotoxin for insect-control. One of the main purposes of this thesis was to construct a chemical-inducible marker-free system for tomato and eliminate the selectable marker gene from Bt transgenic tomato.Another major concern raised by transgenic crops is related to exogenous genes of interest. This concern educes the question of whether crops can be improved by inserting only native DNA into their genomes and to generated cisgenic plants. The virus disease cause severe losses in crop production worldwide. The growing knowledge on eukaryotic initiation factor 4E has provided the novel strategy for genetic engineering for virus resistance. The protein translation mechanism in host is required for virus infection and multiplication. Interfering the process of virus utilizing of the protein translation via genetic engineering not only improves the virus resistance of host plants, but also helps to deepen insights into molecular interaction of plant-virus. So another part of this thesis was to clone the eukaryotic initiation factor 4E from tomato and pepper, and regulate the elF4E expression in tomato and pepper via sense and RNAi strategy. This would not only improve virus resistance in plants but also help to elucidate the function of elF4E in plant virus interaction.Main experiments were carried out as follows:1 A chemical-inducible auto-excision marker free vector system, p35C, was constructed with Cre/loxP site-specific recombination system and an inducible expression system XVE. In vector p35C, three elements including the transactivator XVE, cre and the coding sequence of the neomycin transferaseⅡwere located between the two direct repeats of loxP. XVE was driven by cauliflower mosaic virus 35S promoter upstream of loxP. Cre was driven byβ-estradiol-inducible promoter. Downstream of the next loxP was the crylAc. Upon induction byβ-estradiol, the XVE was activated, and drive the cre expression and DNA recombination between two loxP sites, leading to excision ere, nptll and XVE. Only one intact loxP site would be left and cryIAc would be put directly downstream the CaMV 35S promoter. In the vector p35G, the gfp serves as the trait gene for detect maker gene excision by visualizing green fluorescence.2 Agrobacterium-mediated transformation was carried out on tomato (ZS5) transformation with p35G. Among 19 kanamycin resistant shoots, 2 shoots exhibited green fluorescence, indicating the suitability of this vector for tomato transformation. CrylAc was introduced into ZS5 tomato via p35C, and kanamycin resistant shoots were obtained. The shoots were transferred to inductive rooting media containing 2μMβ-estradiol. Trangenic tomato with single transgene locus was selected for further analysis. T1 progeny analysis was carried out, showing that Cre/loxP-mediated DNA recombination occurrence was 12%～39%, and complete recombination frequency was 8%～30%. Sequence analysis of the recombination region showed the precise and complete recombination at the loxP sites. But in some transgenic lines, e. g. C4 had incomplete recombination. In addition, the attP site fromλbacteriophage was constructed on both sides of nptⅡand introduced into tomato genome for investigation of the recombination occurrence. However, the results showed that the recombination did not happen either in primary transformants or T1 progeny probably because of absence of transformation boost sequence.3 Northern blot analysis was carried out to detect crylAc expression level in transgenic tomato with DNA recombination. And results showed that the cryIAc was significantly transcripted in all transgenic lines tested.4 The CryIAc content in leaves and fruit were assayed by ELISA, and the content was 3500 ng g-1 FW～6300 ng g-1 FW in leaves and 2400 ng g-1 FW～4400 ng g-1 FW in fruits. In vitro insect assay showed that the transgenic lines were conferred improved resistance to cotton bollworm (Helicoverpa armigera Hubner) larva with resistance index between 80%-90%, and insect adjusted mortality was 70%-90%.5 Eukaryotic initiation factor 4E (eIF4E) from tomato ZS5 and pepper Yolo Y was cloned. Tomato elF4E had three nucleotide differences from the reported gene, and pepper eIF4E sequence was the same as the reported gene.6 Sense construct for pepper eIF4E (pCA4S) and RNAi construct for tomato eIF4E (pLE4D) was respectively constructed.7 Agrobacterium-mediated transformation was carried out on tomato (ZS5) with pCA4S and pLE4D respectively. Thirteen kanamycin resistant plants were obtained. PCR and Southern blot analysis showed the integration of the exogenous gene in tomato genome, pCA4S was also used for pepper transformation, and PCR analysis provided the preliminary confirmation of exogenous gene integration in 4 regenerated plants. Because the trait gene, eIF4E, used in this study was from tomato and pepper's own genome, we called the genetically modified plants obtaind as cisgenic plants, although they still contain non-plant-native genes.8 Semi-quantitative RT-PCR was carried out to analysis eIF4E expression in cisgenic plants. Overexpression was observed in cisgenic plants with pCA4S, while gene silence was detected in cisgenic plants with pLE4D.9 Cisgenic tomato plants were challenged with PVY and CMV through rub-inoculation. Through the symptom investigation and semi-quantitative RT-PCR of virus RNA, it was showed that two kinds of cisgenic plants were conferred improved virus resistance. And the cisgenic plants with pLE4D showed more resistance than that with pCA4S. As to different virus, cisgenic plants showed more resistance to PVY than to CMV.10 Cisgenic pepper plants with pCA4S were challenged with PVY and CMV through rub-inoculation. It was shown that cisgenic plants were conferred improved resistance to virus.
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