| Selectable markers based on antibiotic or herbicide resistance have been widely used for selection of transgenic plants in plant transformation, but their potential risks to the environment and human health limited their application as biosafe markers. Selectable marker system using PMI gene encoding to phosphomanose-isomerase (PMI) is such an available candidate of non-resistance marker system. PMI exists widely in bacteria, yeast and animals. However, most plants, excepting to xylocinnamomum, legumes and several other plants, have no PMI gene.Global outbreak of bird flu not only leads to economic loss, but also threat human health seriously in many countries. AIV HA is the major antigen protein. Escherichia coli heat-labile enterotoxin B subunit (LTB) could stimulate ntestinal epithelial cells to enhance immunopotency. In this study, an effective plant expression vector pCAMBIA1302-PMI-MHA-MLTB-MARs harboring PMI selective marker gene, MHA gene, MLTB gene, and MARs was constructed, and then transferred Lotus corniculatus by Agrobacterium tumefacien-mediated method. Results of the PCR, RT-PCR and southern-blotting to the shoots and regenerated plants obtained indicated that target genes were successfully expressed in Lotus corniculatus. Major results obtained are as follows:1. The sensitivity of Lotus explants to mannose was analyzed by exposing explants to different concentrations of mannose and sucrose. The effect of various combinations of sucrose and mannose on Lotus cotyledons explants was used to generate a dose-response curve of the shoot fresh weight after 30 days. The presence of mannose (5–30 g/L) decreased shoot fresh weight. At 20 g/L mannose, there was an average 80% decrease in shoot fresh weight. Mannose at higher concentrations decreased the numbers of shoot at the uniform concentration of sucrose. For the rooting response, Lotus seeds were sown on medium with various concentrations of mannose. After 2 weeks, there was a retardation germination percentage and root length in the media containing increasing concentrations of mannose. Beyond 15 g /L level of mannose did not cause rooting. We established the mannose concentration based on all data.2. The PMI coding sequence was cloned by PCR from pNOV2804. The hygromycin phosphotransferase gene of pCAMBIA1302 was removed by XhoI digestion and replaced with the PMI gene, then was transferred into Lotus corniculatus"Leo"by Agrobacterium tumefaciens. PCR of the shoots indicated that shoots had been transformed. RT-PCR, Southern-blotting and Chlorphenol red assay of selected regenerated plants further confirmed integration of the transgene into the Lotus genome. These positive results suggest that the PMI/mannose selection system, which is free from antibiotic resistance marker genes, could be used for Lotus corniculatus Agrobacterium-mediated transformation. In this study, we achived a transformation efficiency of 23%.3. By genetic recombination technology, the artificially modified gene of avian influenza hemagglutinin MHA and Escherichia coli heat-labile enterotoxin B subunit MLTB were amplified by PCR from pMD-T-MHA and pMD-T-MLTB. Based on the vector pCAMBIA1302, we constructed a fused high effective plant expression vectors: pCAMBIA1302-PMI-MHA-MLTB-MARs (plant expression vector pCAMBIA1302, PMI, nuclear matrix attachment region MAR RB7, CaMV35S promoter, MHA gene, MLTB, NOS terminator).4. The pCAMBIA1302-PMI-MHA-MLTB-MARs was transferred into L. corniculatus explants. PCR of the shoots indicated that shoots had been transformed. RT-PCR, Southern-blotting and Chlorphenol red assay of selected regenerated plants further confirmed integration of the transgene into the Lotus genome. The results indicated that the avian influenza hemagglutinin gene and Escherichia coli heat-labile enterotoxin B subunit were successfully expressed in Lotus corniculatus. We obtained 37 lines of positive transgenic Lotus corniculatus which were infected by vector pCAMBIA1302-PMI-MHA-MLTB-MARs. |
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