| Four transfer Bt gene insect-resistance cotton varieties had been used in this experiment. The study concentrated on the DNA extraction method, PCR reaction system, RAPD reaction system and SSR reaction system,discussing the method for identification of transgenic cottonseed and testing transgenic cottonseed purity compared with the traditional method using kanamycin. The results were the base for the identification of transgenic cottonseed and testing transgenic cottonseed purity in the future. The results were as followed: 1. Identification of transgenic cottonseed using kanamycin We have tested the transgenic cottonseed purity with kanamycin method. The samples were 89% and 86% separately by kanamycin testing. Comparing with the result using PCR testing, we have found two results are consistent. The result using kanamycin was low. Compared with PCR, kanamycin method has some shortages such as long period, costing much work. PCR testing is simple, celerity and delicate. Testing is not restricted with time and sampling parts. The result of the method is credible. 2. Study on isolation of cotton genomic DNA The result of extracting cottonseed DNA at room temperature was better. This method was different from the old method. We have removed the step of purity and the result was ideal. Extract solution was only added to SDS without EDTA and β-mercaptoethanol. The preparation of extract solution was simple. The procedure is followed: ①Grinded the seed sample with normal temperature and transfer the powdered seed to a 1.5m1 tube.② Added 10 times (ul/mg) preheated extraction buffer (100 mM Tris-HCl, 1.5% SDS) to the sample, incubated the mixture at 70℃ 10-50min in the water bath with periodic shaking.③Added equal volume Phenol-Chlorform at room temperature and mixed by inverting the tubes for 1-2 min. Centrifuged for 10 min at 8000rpm at room temperature to separate the phases. The supernatant was used to amplification as DNA template. An efficient procedure had been developed for DNA extraction from the cottonseed based on PCR. The steps of DNA extraction in the study were simple. Time of this procedure was short. As a result, the probability of identification of transgenic cottonseed using PCR was increased rapidly. 3. PCR reaction system The pre-protocol was improved in order to shorten time and amplify specify. A better protocol had been established with the unpurified DNA extraction liquid. Cycle parameters: pre-denature 2min at 94℃,denature 40s at 94℃,anneal 40s at 38℃, extend 50s at 72℃,30 cycles, extend 5min at 72℃.Amplification system: 2.5u1 10×Buffer, 1.5u1 Mg2+ (25mM), lul dNTPs(2.5mM), 0.2u1 TaqE(5U), 0.5ul primer(20pmo1/ul),1 u1 DNA about 60ng, H2O 17.8u1, total volume 25u1. We have tested the transgenic cottonseed purity with PCR method. The samples were 94% and 92% separately by PCR testing. 4. RAPD reaction system All factors affected RAPD reaction were optimized in this study. A better RAPD protocol had been established with the unpurified DNA extraction liquid. Cycle parameters: pre-denature 3min at 94℃,denature 40s at 94℃,anneal 40s at 38℃, extend 60s at 72℃,40 cycles, extend 8min at 72℃.Amplification system: 2u1 10×Buffer, 2.5u1 Mg2+ (25mM), l.8ul dNTPs(2.5mM) , 0.2u1 TaqE(5U), 0.5ul primer(20pmo1/ul),unpurified DNA extraction liquid dilute 10 times , total volume 25u1. In the future, constructing fingerprints map, identification of transgenic cottonseed and testing transgenic cottonseed purity using RAPD will be based on the result. 5. SSR reaction system PCR reaction and detection method of DNA polymorphism on SSR was optimized in this study. The result showed that the best annealing temperature was 55℃ and the best annealing time was 45 seconds. According to our results, for amplification products of SSR polypropylene gel electrophoresis and silver staining was better than agarose gel electrophoresis and ethidium bromide staining. Amount of 3ul amplified product added on gel wells was found enough. The best electrophoresis time was 1.5 hour and 2 hour at 6% and...
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