| In this study, we utilized two-dimensional electrophoresis to examine changes in the total proteins and membrane proteins from the Trichoplusia ni cell line BT1-TN-5B1 exhibiting resistance to Bt Cry 1 Ac endotoxin, and the mRNA changes was examined by mRNA differential display. In addition, we have cloned and sequenced three cDNA fragments of aminopeptidase N family which served as important receptors of Bt Cry 1 Ac. Our results are as following:(1) 707+25 protein dots (n=3) were detected on the resistance cell's proteomic2-DE map, and 637+19 (n=3) on the susceptible cell's proteomic 2-DE map with Melanie Viewer II software.(2) The 2-DE maps between the resistant and susceptible BTl-TN-5Bl's proteomic have remarkable difference. Eleven different dots have been shown. The MW/pI of them are 98.5/4.5, 88.7/6.5, 65.5/4.8, 39.2/4.7, 38.8/5.4, 35.5/6.7,28.5/8.2, 22.5/8.7,21.8/8.3,21.1/7.0 and 21.1/6.4.(3) One differential dot on susceptible cell's 2-DE map was analysed using the peptide mass fingerprint technology, the result showed that the protein was similar to a periplasmic protein, so it appeared that it may be one kind of membrane protein.(4) 290+10 protein dots (n=3) were detected on the resistance cell's membrane proteins 2-DE map, and 295+10 (n=3) on the susceptible cell's membrane proteins 2-DE map with Melanie Viewer II software.(5) The 2-DE maps between the resistant and susceptible BTl-TN-5Bl's membrane proteins have remarkable difference. Ten different dots have been shown. The MW/pI of them are 66.0/6.8, 34.6/5.8, 31.5/6.8, 31.0/7.1, 31.4/7.3, 28.5/7.1, 27.0/6.5, 20.7/5.9, 20.0/5.8, 14.4/6.5.(6) The results of DDRT-PCR shown that there are transcriptional difference between resistant cell and the susceptible cell.(7) Five differential ESTs have been identified between resistant cell and the susceptible cell, three of them from the susceptible cell, named them S1(CF322415) S2(CF322416) and S3(CF322417), and the other two from the resistant cell named them R1(CF322413), R2(CF322414).(8) The deduced amino sequence from Rl was similar to phosphoenolpyruvate carboxykinase, so we suppose that the gene corresponding to it encode one kind of phosphoenolpyruvate carboxykinase.(9) The deduced amino sequence from R2 was similar to mucin, they are the important communent of cell membrane, so we suppose that the gene corresponding to it encode one kind of mucin.(10) We have cloned three aminopeptidase N cDNA fragment, named them APNS1(CD8O9324) , APNS2(CD809325) and APNS3(CD809326) respectively, and they represent three kind of aminopeptidase N respectively.(11) We have identified that all of the three aminopeptidase genes expressed both the susceptible cell and resistant cell.Some previous reports have shown that the mechanism of resistance in insects to Bt is multifaceted (complex). Basing on the results of our studies, we suppose that some possible resistance mechanisms to Cry 1 Ac in BT1-TN-5B1 are as following:(1) Our studies has identified some different proteins and some differential expressing genes between the resistant cell and susceptible cell, So, the results of our studies support the hypothesis that the resistance to Cry 1 Ac in BTl-TN-5Blis multifaceted.(2) The membrane proteins changes between the resistant cell and susceptible cell have been identified. The membrane is the target of Bt toxin, so we suppose that the changes of many membrane proteins are the most important molecular basis of the resistance BT1-TN-5B1 to CrylAc.(3) The resistance cell contained phosphoenolpyruvate carboxykinase which can not been detected in the susceptible cell, so we suppose that the resistantto CrylAc toxin in BT1-TN-5B1 may has some relation to oxidative metabolism.Not only did this study provided a solid foundation for the further investigation on the resistant mechanism related to CrylAc toxin, but also it was beneficial to realize the mechanism of the resistance to Bt... |
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