| Floristic disease is one of the major limited factors in crop production. Breeding novel varieties is the key countermeasure to meet this complexity by using antifungal and antibacterial resource at present. However, which can not respond to rapid aberrance of pathogen besides limited resources and longer cultivated periods. Development of molecular biology technique, especial for genetic engineering, presents a novel approach to conquering diseases and enhancing crop production.Ribosome inactivating protein (RIP) and non-specific lipid transfer proteins (nsLTPs) are frequently utilized to resist disease in genetic engineering. They had been purified from Leonurus japonicus seed, corresponding genes were copied and high efficient expression vectors harboring plant were constructed by Xingyong Yang, which named RIP, Lh-L and Lh-S, respectively. In this paper, they are introduced into Nicotiana tabacum cv by agrobacterium mediated transformation, leaf dish as explants. Transformed plants are confirmed and resistant levels are analyzed. Results are as following:1. Transferring foreign genes to tobacco genomeLh-L, Lh-S and RIP genes were respectively transfer into tobacco genome by agrobacterium mediated transformation, Leaf dish as host. Fifty seedlings against NPTII are obtained for each gene by using different transformation.2. Identification of transgenic plantTransformed plants were first stained by GUS solution, showing that foreign gene had been introduced and regenerated into tobacco genomes. They were further confirmed by PCR assay. GUS positive plants could be amplified a stable band with molecular weight conforming to positive control; however GUS negative plant could not. RT-PCR assay revealed that foreign genes could steadily transcribe in transgenic tobacco plants.3. Copy number and regenerated loci identification of foreign genesThree transgenic plant lines were utilized to identify foreign gene copy numbers and regenerated locus for each foreign gene. T0 seeds were stained with GUS solution just after germination for about three hundreds to seven hundreds. GUS positive and GUS negative rate was analysed by xc2, showing that their inherited rate has no remarkable difference with 3:1. This revealed that foreign gene occupied tobacco genomes by single locus. Considering single band amplified by PCR, copy number of gene must also be single.4. Concentration of pathogeny utilizing for examination of disease resistanceSpore of Alternaria alternata was filtrated from suspension using monolayer filter paper; this was feasible compared with other methods. Spore was diluted for five kinds of concentrations; each increased ten fold from 1×10~3 to 1×10~7 conidia per milliliter. Assays showed that the available disposal was 1×10~6 conidia per milliliter. As for living plants, preferable inoculated concentration of pseudomonas solanacearum was 0.8OD by performing disease-resisted experiment; four disposals were 0.2OD, 0.5OD, 0.8OD and 1.0 OD.5. Disease resistance analysis for transgenic plant5.1 Inoculating Alternaria alternata to separate leaf of To- transgenic plantTo-transgenic plants were inoculated with Alternaria alternata in greenhouse. Comparison them with wild-type control showed that resistance enhanced distinctively in transgenic plants. Disease indexes were 27.66%, 40.77% and 30.36% respectively corresponding to transgenic plants with Lh-L, Lh-S and RIP. Then conclusion could be got that Lh-L gene was most suitable for genetic engineering applied to resist plant disease among three genes.5.2 Inoculating Pseudomanas solanacearum to living T0- transgenic plantRoot inoculation of plantlets was utilized to assess transgenic plant resistance against Pseudomanas solanacearum. Result demonstrated that resistance was enhanced and lead to remarkable level for all transgenic plants compared with non-transgenic plants. Comparation of each transgenic plant lines suggested that Lh-L-1 and Lh-L-2 were most preferable in resistance and got to remarkable level, next choice were Lh-L-3, RIPS and Lh-S-1, RIP-I and RIP-2 were prone to Lh-S-2 and Lh-S-3, and the later were the worst in this assay.5.3 Inoculating Pseudomanas solanacearum to living T1- transgenic plantT1 transgenic plants were inoculated with Pseudomanas solanacearum in greenhouse and resistance was further analysed for three genes. Result confirmed the resistant results from To-transgenic plants, and all plant lines were preferable remarkably to wild-type control. Disease index of RIP was lower than Lh-S, and Lh-L was lowest among three applied genes. This shows that antibacterial effect can inherited to offspring in transgenic plant. Comparation with RT-PCR results showed that resistance increased with foreign genes transcription at primary, however after a certain...
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