| In the paper, the technique for testing purity and identification of resistance to cotton boll worm of Bt-transgenic cotton is probed into by methods of field experiment , chamber bioassay, biochemical and immune determination. The main results are summarized.1. Technique for testing seeds purity of Bt-transgenic cotton using marker geneAccording to beta-glucuronidase (GUS) gene and nomycin phosphotraseferse II (NPTII) gene in the exogenous gene transformed into Bt-transgenic cotton cultivar "Guo-Kang No.l". Three methods of testing the purity of commercial seed of Bt-transgenic cotton are established: the method of chemical GUS assay, the method of Kanamycin(Km) medium and the method of Km moisteining. The accurate rates all get to 98%. The accurate rates also get to 98%(compared to the result of bioassay) in testing the Bt-transgenic cotton original group "20-1". Otherwise, there is only NPT II gene in "Bao-ling mian 32B" introduced from America. The accurate rates get to 98% in testing "Bao-ling mian 32B" using the method Km medium and Km moisteining. The Bt-transgenic cotton "Bao-Ling mian 32B". So, the three methods can be put to use in testing seeds purity of Bt-transgenic cotton and the corresponding methods are suitable to every kind of Bt-transgenic plant cultivar whose genome contained GUS gene and(or) NPT II gene.2. Dot immunogold filtration assay testing and identifying Bt-transgenic cottonThe mixture of spores and crystals from Bacillus thuringiensis strain HD-1 is first solubized with alkaline buffer and precipitated again with acid , then followed by trypsinization, ammonium sulfate precipitation and get filtration chromatography, finally, two kinds of polypeptide with a molecular weight of about 66.2KD estimated by SDS-PAGE were purified . The purified polypeptideshowing toxin activity to Heloicoverpy armigera by ingestion is proved to be Bt toxinrCry I A(b) and Cry I A(c).Using Bt toxin as antigen and polycolnal antibody against Bt toxin as antibody, the antigen-sandwich immunofiltration assay is developed. Furthermore, the dot immunogold filtration assay(DIGFA) is established by information being amplified because of biotin-avidin system(BAS) and the kit for detecting Bt toxin by dot immunofiltration assay is preliminarily developed.In this trial the sodium citrate reduction method is used to manufacture 15nm colloidal gold. The satisfactory result can be achieved using magnetic agitating heater to prepare colloidal gold.When the polyclonal antibody and streptavidin are labeled to 15nm colloidal gold, the optimal protein protection amount is 9ug/ml and 16ug/ml respectively and the optimal protein labeling amount is 11.7ug/ml and 20.8ug/ml respectively. The result is not satisfied using immunogold antibody as immunogold probe and the result is satisfied by developing the antigen-sandwich immunofiltration assay using biotin labeling antibody and using immunogold streptavidin as immunogold probe.The process of assembling device of the kit includes the steps as follows: preparing membranes, coating, marking, blocking, washing and assembling. Further study is carried out to optimize different reagent applying amount. The membrane used is Nitrocellulose(NC) membrane from Amersham Company. The antibody coating the membrane is concentrated after the membrane being soaked in 80% alcohol then drying 5 hours in room temperature or 3 hour in 37癈 constant temperature box. The buffer diluting antibody is PH8.4 0.005M borate buffer. The antibody concentration coating the membrane is 62.5ug/ml. lul. The optimal blocking reagent is l%BSA+0.5%Tween-20, 20ul, then the membrane is laid aside for 0.5h. the dilution of the biotin labeling antibody is 1:300,lul. Immunogold probe have something to do with detecting quality while amount the dilution of immunogold streptavidin is 1:3, 3ul with clear dot and lower background. In the dot for controlling quality, the antigen concentration coating the membrane is 125ng/ml, lul. The buffer diluting antigen is pH9.0 0.005Mbora...
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