| Melon is one of the unique fruits species in Xinjiang, and it can produce preferableeconomic benefits. The occurrence of melons downy mildew has an immense impacton grain yield and quality of melon. Plant gene engineering methods can be appliedto improve crop germplasm traits. The reports on downy mildew resistance gene bothat home and abroad became more useful. The relative resistance of At2gene is a kindof enzyme, which involved in plant in the process of metabolism of photorespirationserine: hydroxyl pyruvate transaminase reaction. SGT enzyme and downstreamenzymes involved GO glyoxalic acid oxidase activity can be the direct reaction oftransgenic melon resistance to downy mildew. Double T-DNA expression vectorsystem can not only realize the successful transformation of gene, also can use tochoose the safety of genetically modified crops through the offspring the individualgenetic recombination and chromosome screening. Therefore, this method is widelyused in various crops safe transgenic research.This experiment adopted double T-DNA expression vector to transmitted downymildew resistance gene At2into melon "Queen", which was successfully conductedby preliminary laboratory research. We evaluated T0generation of transgenic plantsby molecular level in the positive transformation of individual screening. Wecollected the leaves from untransformed "queen” and transgenic plant before andafter vaccination, and further analyses the enzyme quantity of leaf about GO, AGT,SGT, CAT and SOD activities. We also detected the contents of hydrogen peroxideand oxygen free radicals in the leaves. Through downy mildew spores inoculated invitro respectively, two transgenic melon, transgenic melon A (nptⅡ) T0generation,transgenic melon B (T-DNA) T0generation, of which transgenic leaves resistanceability were analyses.The main results as following:(1) Transformation experiments2069explants were transformed, the conversion ratewas1.11%of total of23transformed individual, At2and bar gene conversionrate was0.43%. Successful transformation positive individuals by PCR and RT-PCR assay does success into in the genome of melon. q RT-PCR analysisconfirmed At2gene expression quantity levels were indeed increased intransgenic melon leaves. Prior to the laboratory for genetically modified melonobtained (nptⅡ) conducted field hardening and harvested the seeds, and its T1generation plants for further molecular test. The results showed that the At2intogene can be stably transferred, and genetic expression of the gene in the offspringseparation was strictly in accord with Mendelian segregation ratio.(2) Physiological and biochemical analysis were detected from the leaves of controlmelon, transgenic melon (nptⅡ) and transgenic melon B (T)-DNA and downymildew inoculated plants. The results showed that the transgenic melon A (nptⅡ)At2gene expression increases after inoculation, this was in line with SGT changeof enzymatic activity and CAT expression quantity was markedly reduced, SODvalue compared also decreased dramatically between before and after vaccination,the contents of H2O2and oxygen free radical increased after vaccination.Transgenic plant is under a state of stress. The express of At2from transgenicmelon B (T-DNA) was no significant difference before and after vaccination,SGT enzyme activity before and after inoculation did not increase, CATexpression quantity descended obviously, and H2O2and oxygen free radicalcontent compared with no significant differences before and after inoculation.(3) After that inoculated with the downy mildew spores, It showed that transgenicmelon A(nptⅡ) really got a certain resistance against downy mildew. Vaccinationafter10d, other pathogenic bacteria appeared on the medium and the transgenicplants Blade was still in good condition. Therefore, At2gene may have resistanceto other pathogenic bacteria. |
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