摘要
以对Bt Cry1Ac毒素十分敏感的BT1-TN-581细胞和经过Cry1Ac筛选了83代的同源抗性细胞为实验材料，采用双向电泳和mRNA差异显示技术分别从蛋白质水平和转录水平较系统的揭示了两者之间的差异，另外还通过设计简并引物，对作为Cry1Ac的重要受体之一的氨肽酶N家族成员进行了cDNA片段克隆。实验结果如下：
(1) 得到了抗性细胞和敏感细胞总蛋白的2-DE图谱，通过Melanie Viewer Ⅱ软件分析，在抗性细胞的总蛋白2-DE图谱上检测到的平均蛋白质点数为707±25(n=3)个，在敏感细胞的总蛋白2-DE图谱上检测到的平均蛋白质点数为637±19(n=3)个。
(2) 两者总蛋白的2-DE图谱存在显著差异，初步确定了在两者总蛋白2-DE图谱上存在显著差异的11个蛋白质点的MW／pI，分别为：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、21.1／6.4。
(3) 对一种敏感细胞特有的蛋白质点进行了肽质指纹图谱分析，同源性收索结果表明它与胞质外周蛋白有较高相似性，推测它为一种膜蛋白。
(4) 得到了抗性细胞和敏感细胞的膜蛋白2-DE图谱，通过Melanie Viewer Ⅱ软件分析，在抗性细胞的膜蛋白2-DE图谱上检测到的平均蛋白质点数为290±10(n=3)个，在敏感细胞的膜蛋白2-DE图谱上检测到的平均蛋白质点数为295±10(n=3)个。
(5) 抗性细胞和敏感细胞的膜蛋白2-DE图谱存在显著差异，初步确定了膜蛋白2-DE图谱上存在差异的10蛋白质点的MW／pI，分别为：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) mRNA差异显示结果表明抗性细胞品系和敏感细胞品系在转录水平上存在差异。
(7) 初步鉴定了5个在两种细胞之间存在差异的ESTs序列，三种为敏感细胞特有分别命名为：S1(CF322415)、S2(CF322416)、S3(CF322417)，
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两种为抗性细胞特有，分别命名为:RI(CF322413)、R2(CF322414)。
(8)根据Rl序列推导的氨基酸序列与磷酸烯醇式丙酮酸梭激酶与较高
同源性，推测它所对应的基因可能编码磷酸烯醇式丙酮酸梭激酶。
〔9)根据R2序列推导的氨基酸序列与糖蛋白和粘蛋白类有一定的同源
性，推测它所对应的基因可能编码一种糖蛋白或粘蛋白。
(10)获得了3条分别代表3种氨肤酶N基因的ESTs片断，分别命名
为:APNSI(CD809324)、APNSZ(CD809325)、APNS3(CD809326)。
(川初步确定本实验所鉴定的三种氨肤酶基因在两种细胞群体中都有
表达。
现阶段的诸多研究都表明昆虫对Bt产生抗性的机制十分复杂，鉴于
本实验所获得的结果，我们推测BTI~TN一5BI对Cry1A。抗性产生的分子
基础和可能的机制如下:
(l)本实验结果表明多种蛋白质在抗性细胞和敏感细胞中存在差异，
多种基因在两种细胞中存在差异表达，这些结果表明BTI~TN一5BI对
cry1Ac抗性是多因素控制的。
(2)多方面的信息都表明抗性细胞和敏感细胞的膜蛋白组分存在差
异，而膜蛋白是Bt Cry1Ac作用于细胞的直接靶标，因此我们推测膜蛋白
的改变可能是BTI~TN一5BI对Cry1A。产生抗性的重要分子基础。
(3)我们在实验中证实磷酸烯醇式丙酮酸梭激酶在抗性细胞和敏感
细胞中差异表达，而磷酸烯醇式丙酮酸梭激酶是三梭酸循环回补反应的
重要酶类，因此推测BTI一TN一SBI对Cry1Ac抗性的产生可能还涉及细胞
能量代谢。
本实验为进一步研究BTI一T’N一5BI对Cry1Ac抗性的分子机理奠定了
基础，也为Bt抗性机理的研究工作提供了一定的依据。
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 resistant
to 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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