| PemG1activator protein from Magnaporthe grisea is able to promote seeds germination and seedlings growth, and also induce cotton and tobacco resistance against plant disease. Cotton bollworm (Helicoverpa armigera) is a very harmful agricultural pests, because of its feeding host is relatively wide, causing huge economic losses every year in China and around the world, but long-term use of chemical pesticides will cause bollworm resistance pesticide, and also pollute the environment. Maybe inhibiting its molting is a good method which can control the number of the pests.In this study the transcription factors of regulating molting of Helicoverpa armigera were choosed as the target gene, which regulate the related gene expression in the process of molting. In order to explore the pemG1protein’s function of induced resistance in plants and promote the growth function of plants, RNA interference mediated was applied to inhibit of gene silencing transcription factor and regulate the expression of molting. The metamorphosis can impede the normal development of cotton bollworm, and Regulate population number, it provide a new way for the control of cotton bollworm.In this paper, the plant expression vector pCAMBIA2300-Ubi-pemG1-Ocs which include of target gene pemG1was constructed. By using Agrobacterium-mediated transformation of the shoot tip and hypocotyl of the cotton cultivar Coker312and Chinese cotton24, after series of tissue culture and the process of transformation, to obtain resistant plants, and postive plants were obtained by PCR, sequencing, RT-PCR, Southern detection. The positive strains were inoculated with Fusarium oxysporum, The result showed the transgenic cotton resistance to Fusarium wilt of cotton.And the other gene from bollworm molt containing transcription factors of HaHR3RNA interference vector,which named pCAMBIA-RNAi-HaHR3, was transferred into Nicotiana tobacum cv.Samsun NN leaf discs by Agrobacterium-mediated method, according to the relevant molecular detection, of plants within the gene integration, transcription, analysis of transgenic insect-resistant traits were identified, it provide a platform for application of RNA interference-mediated control to the number of Helicoverpa armigera.The main results of this study are as follows:1. Construction of the plant expression vectors pCAMBIA2300-pemG1and pCAMBIA2300-RNAi-HaHR3containing the constitutive promoter of35s of the cauliflower mosaic virus, octopine synthase terminator system and kanamycin-resistant gene nptⅡ (neomycin phosphotransfers Ⅱ). DNA constructs were verified by restriction enzyme digestion and sequencing, then the plant expression vectors were transformed into Agrobacterium tumefaciens strain LBA4404by freeze-thaw method. This method was used for Agrobacterium-mediated transformation of cotton and tobacco.2. Transferred the vector pCAMBIA2300-pemG1, pCAMBIA2300-RNAi-HaHR3via Agrobacterium tumefaciens respectively into hypocotyls of cotton and its shoot tip, through the combination of the antibiotic and screening, grope for the culture medium, the composition of hormone and regulation, obtained the final kanamycin resistant plants.3. By Agrobacterium-mediated transfer PCAMBIA23002300-RNAi-HaHR3into tobacco leaf and obtained the resistant plants, and with the T2generation.4. Transgenic plants were confirmed to be the positive ones by polymerase chain reaction (PCR) and sequencing.The pemG1gene and HaHR3integration was further confirmed by Southern blot. The transgene transcription was verified by reverse transcription polymerase chain reaction (RT-PCR).5. Comparative and analysis of different hormones in different concentrations of BAP and NAA combination to the growth of budding, the results show the combination of0.1mg/L of BAP and NAA is best for bud induction.6. Ananisis of the relevant disease resistant and agronomic trait by transgen pemG1into cotton, the result shows the transgenic cotton has good preventive effect against Fusarium wilt. By bioassay of transgene HaHR3tobacco we can find it has a good inhibitory action to the growth of cotton bollworm... |
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