| The serine/threonine protein kinase gene (GbSTK) and the glutathione s-transferase gene(GhGST) related to Verticillium wilt resistance were cloned from island cotton variety Pima90-53 and resistant upland cotton variety Jimian20, respectively. The overexpression vector pGN-GbSTK and pBI121-GhGST were constructed and transformed into Arabidopsis thaliana and tobacco in our laboratory. In the study, the expression analysis of different times and different varieties was completed by real time-PCR technology. The two genes were transformed into cotton using Agrobacterium-mediated method and pollen tube channel method. The different expression of GbSTK in T3 transgenic Arabidopsis thaliana induced by Verticillium dahliae was researched. The main resultswere as follows:1. The real time-PCR experiments showed that the expression of GbSTK and GhGST in cotton cultivars with different resistance have significant differences. The strongest expression was observed in resistant variety Pima90-53, followed by Jimian20, and the weakest expression was observed in susceptible variety Han208. The experiments indicated that GbSTK and GhGST genes may be related to induction of Verticillium dahliae.2. The real time-PCR experiments showed that different expression of GbSTK was observed in T3 transgenic pCamGbSTK::GFP Arabidopsis induced by Verticillium dahliae. GbSTK had high level expression after 24h Verticillium dahliae stress, the expression amount is about four times that of 0h. The expression of GbSTK had not been detected in wild type Arabidopsis. The results showed that GbSTK involved the reaction against verticillium wilt in the transgenic Arabidopsis.3. The shoot apex of Han208 (Gossypium hirsutum) was selected for regeneation and transformation. The effects of gelling agents, plant growth regulators and antibiotics were investigated on regeneration and root-inducing rate, and the effects of Agrobacterium concentration, infection time, the time and the temperature of co-culture on the transformation rate of shoot apex were studied by orthogonal design. The results showed that adventitious buds of shoot apex were efficiently induced by 0.1mg/L 6-BA +0.1mg/L IAA, and roots of shoot apex were efficiently induced by 1/2MSB +0.1mg /L IAA+0.1 mg/L NAA with phytagel as gelling agents. The treatment on shoot apex by 0.9 of OD600 value of agrobacterium suspesion, infection time for 25min, and co-culturing for 48h in 22°C could obtain the highest transformation rate at 75 mg/L Kan. Using the genetic transformation system, GhGST gene was transformed into cotton variety Han208, and 3 genetically modified plants were obtained by PCR detection.4. The effects of cultivation mode, training time, treatment of hypocotyl, and agrobacterium type were investigated on callus induction. The results showed that the highest resistance callus induction rate was obtained with hypocotyls cut from 3 days of dark training sterile which is infected by LBA4404. The GhGST was transformed into cotton variety Jimian19 and Coker312, the resistance callus was obtained and the positive callus was also detected by PCR.5. GhGST was transformed into susceptible variety Jimian11 and CRI 8 by pollen tube channel method. Five T0 plants of Jimian11 and two of CRI8 were obtained by PCR detection. |
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