摘要
野桑蚕(Bombyx mandarina)和家蚕(Bombyx mori)为鳞翅目昆虫。对野桑蚕和家蚕进行拟除虫菊酯抗性的比较研究,将为鳞翅目害虫的防治和家蚕抗性育种提供重要的理论依据。拟除虫菊酯的作用靶标是神经轴突上的钠离子通道蛋白(sodium channel protein),而细胞色素P450第九家族的多个基因与昆虫对拟除虫菊酯抗性有关。本文测定了溴氰菊酯对野桑蚕和家蚕的毒力,进行了抗溴氰菊酯野桑蚕的筛选,克隆了野桑蚕和家蚕的钠离子通道蛋白基因,分析了氯氰菊酯对野桑蚕和家蚕CYP9家族基因的诱导转录,获得的主要结果如下:
1野桑蚕拟除虫菊酯抗药性和钠离子通道蛋白基因的克隆
1.1溴氰菊酯对野桑蚕和家蚕的毒力比较
采用点滴法测定了溴氰菊酯对野桑蚕和家蚕的毒力。吴江野桑蚕(YWJ)、启东野桑蚕(YQD)和苏大野桑蚕(YSD)分别是家蚕品种大造(Dazao)的15.89倍、11.67倍、10.11倍,家蚕品种L11是大造的4.37倍。
1.2野桑蚕对溴氰菊酯抗性品系的筛选
在桑叶育条件下,苏大野桑蚕经连续3代溴氰菊酯筛选,S4代抗药性倍数为16.48倍,S4代LD50 (0.445 ng/头)值较S0代LD50 (0.237 ng/头)提高了0.9倍。在人工饲料饲育条件下,吴江野桑蚕经6代人工饲料育和3代溴氰菊酯筛选,S6代抗药性倍数为18.67倍,S6代LD50 (0.504 ng/头)值较S0代LD50 (0.418 ng/头)值提高了0.2倍。
1.3野桑蚕和家蚕para型钠离子通道蛋白基因结构域Ⅱ的克隆与突变
根据家蚕钠离子通道蛋白基因部分序列(GenBank登录号:EF521818),设计特异引物,通过RT-PCR方法,克隆了编码吴江野桑蚕(YWJ)和家蚕品种大造的钠离子通道蛋白结构域II的cDNA片段。两片段长均为1 135bp ,编码378个氨基酸,没有发现与击倒抗性(knock down resistance,kdr)相关的点突变,但发现2个点突变I164L和V196A,推测可能这两个突变与野桑蚕对溴氰菊酯的抗性有关。
1.4野桑蚕和家蚕para型钠离子通道蛋白基因的克隆与选择性剪切
根据家蚕钠离子通道蛋白基因部分序列(GenBank登录号:BK005824,EF521818),设计引物,分段RT-PCR,克隆了家蚕大造钠离子通道蛋白基因Bmpara(GenBank登录号:EU688970)和吴江野桑蚕钠离子通道蛋白基因Bmmpara。根据克隆的野桑蚕钠离子通道蛋白基因序列和家蚕基因组序列,设计引物,采用PCR方法,克隆到3段野桑蚕基因组序列。序列分析表明,Bmpara的cDNA长为5 555 bp,编码1 851个氨基酸;Bmmpara的cDNA有6种,长度分别为5 555 bp、5 594 bp、5 432 bp、5 522 bp、5 561bp、5 399bp,分别编码1 851、1 864、1 810、1 840、1 853、1 799个氨基酸;3段野桑蚕基因组序列长度分别为1 632 bp、536 bp、3 220 bp。将Bmpara和Bmmpara的cDNA与家蚕基因组Blast分析和野桑蚕基因组片段比对,结果表明,Bmpara有34个外显子和33个内含子,Bmmpara除了与Bmpara有相同结果外,还发现Bmmpara有a(11个氨基酸ENDLGRTKKKK)、b(13个氨基酸GLKAALCGRCVSS)、c(41个氨基酸SLINFVAALCGAGGIQAFKTMRTLRALRPL RAMSRMQGMRV)3个选择性微外元,可能存在6种选择性剪接,构成野桑蚕钠离子通道蛋白不同亚型。
2野桑蚕和家蚕细胞色素P450的诱导与抗药性研究
2.1野桑蚕细胞色素P450氧化酶活性测定及CYP9家族基因的诱导转录
采用酶标板酶活性动力学法检测了氯氰菊酯诱导苏大野桑蚕中肠和脂肪体的PNOD活性,结果表明:氯氰菊酯诱导24 h,脂肪体、中肠PNOD活性分别增加了18.7%、12.2%。根据家蚕CYP9家族基因序列,设计引物,克隆了野桑蚕CYP9A21基因(GenBank登录号:FJ265741)、CYP9A22基因(GenBank登号:EF535806)和CYP9G3基因片断。用半定量RT-PCR方法分析了氯氰菊酯诱导野桑蚕CYP9家族基因的表达水平,结果表明,氯氰菊酯诱导24 h,CYP9A21基因在中肠的mRNA表达量是对照的2.1倍,CYP9A20、CYP9A21、CYP9G3基因在脂肪体的mRNA表达量是对照的1.9、3.5、1.4倍。推测野桑蚕CYP9A21等基因的的过量表达可能导致野桑蚕对氯氰菊酯氧化解毒代谢的增强。
2.2家蚕CYP9家族基因的诱导转录及CYP9A22的克隆与序列分析
用半定量RT-PCR方法分析氯氰菊酯诱导家蚕CYP9家族基因的表达水平,结果表明,氯氰菊酯诱导家蚕24 h, CYP9A20基因的中肠、CYP9A22基因的脂肪体、CYP9G3的脂肪体的mRNA表达量分别是对照的1.3、3.1、1.2倍。与野桑蚕相比,家蚕CYP9家族基因被诱导的mRNA表达量低,推测这可能是家蚕比野桑蚕对拟除虫菊酯更敏感的原因之一。采用RACE策略,克隆了CYP9A22基因(GenBank登录号:EF535804)。该基因cDNA全长1 707 bp,编码区长1 596 bp,编码531个氨基酸,推定的蛋白质分子质量为61 kD,等电点为7.71,与已知的CYP9家族的CYP9A14基因(棉铃虫Helicoverpa armigera)的氨基酸同源性达到62.3%。利用NNPP分析软件预测出转录起始位点,与根据CYP9A22全长cDNA序列推测的结果是一致的。TFSEARCH 1.3软件分析转录因子结合位点的结果显示,转录起始位点上游序列不仅包含启动子的核心结构序列TATA-box和CAAT-box,亦包含多个转录因子结合位点,如GATA-1,CdxA,Dfd等。
Bombyx mandarina and Bombyx mori are lepidopterous insects. The comparative study of resistance to pyrethroid in Bombyx mandarina and Bombyx mori will be of great significance to lepidoptera pests prevention and resistive breeding of Bombyx mori. Action drone of pyrethroid is sodium channel protein located in nerve axone, while several genes of the ninth cytochrome P450 family are related to resistance to pyrethroid in insects. In this study, we measured the toxicity of decamethrin on Bombyx mandarina and Bombyx mori, screened Bombyx mandarina with decamethrin resistance, colned sodium channel protein genes in Bombyx mandarina and Bombyx mori, and investigated the transcription of CYP9 family genes induced by cypermethrin in Bombyx mandarina and Bombyx mori. The main results are summarized as follows:
1 Research on Pyrethroid resistance and sodium channel protein genes clone in Bombyx mandarina
1.1 Comparison of the toxicity of deltamethrin on Bombyx mandarina and Bombyx mori
Drop method was used to determinate the toxicity of deltamethrin on Bombyx mandarina and Bombyx mori. The resistance of Wujiang Bombyx mandarina (YWJ), Qidong Bombyx mandarina (YQD) and Soochow University Bombyx mandarina (YSD) was 15.89, 11.67 and 10.11 times of that of Dazao Bombyx mori, respectively, while the resistance of L11 Bombyx mori was 4.37 times of that of Dazao Bombyx mori.
1.2 Screening of deltamethrin resistant strains of Bombyx mandarina
Deltamethrin was used to screen resistant strains of Bombyx mandarina. YSD fed with mulberry leaves were screened for three generations under deltamethrin treatment continuously. The results showed that the deltamethrin resistance of S4 was 16.48 times of that of Dazao Bombyx mori, and the LD50 value (0.445 ng/larva) in S4 generation increased by 0.9 times in comparison with the LD50 value (0.237 ng/larva) of S0 generation. YWJ fed with artificial diets for six generations were screened for three generations under deltamethrin treatment. The results suggested that the deltamethrin resistance of S6 was 18.67 times of that of Dazao Bombyx mori, and the LD50 value (0.504 ng/larva) in S6 generation increased by 0.2 times compared with the LD50 value (0.418 ng/larva) of S0 generation.
1.3 The cloning and mutation of para sodium channel protein gene structure domainⅡin Bombyx mandarina and Bombyx mori
According to the partial sequence of Bombyx mori sodium channel protein gene (GenBank No. EF521818), the special primer was designed. The cDNA fragments which encoded the sodium channel protein encoding gene structure domainⅡof YWJ and Dazao were cloned by RT-PCR. Sequence analysis showed that the length of the two cDNA fragments was about 1135bp, respectively, which encoded 378 amino acids. No point mutation related to kdr resistance and super-kdr was found, while two amino acid point mutations, namely I164L and V196A, were discovered, which might be relevant to deltamethrin resistance of Bombyx mandarina.
1.4 Cloning and selective splicing of sodium channel protein genes of Bombyx mandarina and Bombyx mori
According to the partial sequence of Bombyx mori sodium channel protein encoding gene (GenBank No. BK005824,EF521818), the special primes were designed. The sodium channel protein genes of Bombyx mori Bmpara (GenBank No. EU688970) and Bombyx mandarina Bmmpara were cloned, respectively, by RT-PCR. According to the sequences of the cloned sodium channel protein genes in Bombyx mandarina and Bombyx mori genome, three Bombyx mandarina genome sequences were cloned by PCR. Sequence analysis indicated that the length of Bmpara cDNA is 5 555 bp, which encoded 1851 amino acids. There are six kinds of Bmmpara cDNA, the lengths of which were 5 555 bp, 5 594 bp, 5 432 bp, 5 522 bp, 5 561 bp, 5 399 bp, respectively, and encoded 1 851, 1 864, 1 810, 1 840, 1 853, 1 799 amino acid, respectively. The lengthes of three Bombyx mandarina genome sequences were 1 632 bp, 536 bp and 3 220 bp, respectively. The Blast anlysis and comparison analysis showed that there were at least 34 extrons and 33 introns in Bmpara and Bmmpara. Moreover, there were three optional micro-exons in Bmmpara,including a with eleven amino acids (ENDLGRTKKKK), b with thirteen amino acids (GLKAALCGRCVSS) and c with forty-one amino acids (SLINFVAALCGAGGIQ AFKTMRTLRALRPLRAMSRMQGMRV), six kinds of selective splicing, which established different subtypes of sodium channel protein of Bombyx mandarina.
2 Inducement of cytochrome P450 and drug resistance research in Bombyx mandarina and Bombyx mori
2.1 P450 oxidase activity determination and induced-transcription of CYP9 family genes in Bombyx mandarina
Enzyme linked immunosorbent assay was used to determine the activity of PNOD in the midgut and fat body of YSD under cypermethrin treatment. Result showed that after 24h inducement, the PNOD activity of fat body and midgut increased by 18.7% and 12.2%, respectively. With the primer designed according to CYP9 family gene of Bombyx mori, CYP9A21 (GenBank No. FJ265741), CYP9A22 (GenBank No. EF535806) and CYP9G3 segments of Bombyx mandarina were cloned, respectively. The expression level of CYP9 family genes of Bombyx mandarina induced by cypermethrin were determined by semi-quantitive RT-PCR. Results indicated that after 24h inducement, the mRNA expression amount of CYP9A21 in midgut is 2.1 times of the control, and the mRNA expression amount of CYP9A20、CYP9A21、CYP9G3 in fat body were 1.9, 3.5, and 1.4 times of the control, respectively. The over expression of these special cypermethrin P450 genes might enhance the oxidation and detoxifcation capacity of Bombyx mandarina to cypermethrin.
2.2 Induced-transcription of CYP9 family gene of Bombyx mori and clone and sequence analysis of CYP9A22
Semi-quantitive RT-PCR was used to analysis expression level of CYP9 family genes in Bombyx mori induced by cypermethrin. Results indicated that after 24h inducement, the mRNA expression amount of CYP9A20 in midgut, CYP9A22 and CYP9G3 in fat body, were 1.3, 3.1, 1.2 times of the control, respectively. Compared with Bombyx mandarina, the mRNA expression amount of CYP9 family genes in Bombyx mori were lower, which partially accounted for the more susceptivity of Bombyx mori to pyrethroid than Bombyx mandarina. CYP9A22 (GenBank No. EF535804) of cytochrome P450 was cloned by RACE. The total length of the cDNA is 1707 bp, and the length of encoding region is 1596bp encoding five hundred and thirty-one amino acids. The deduced molecule weight of the protein is 61KD with pI 7.71, and the amino acid homology between CYP9A22 and CYP9A14 of CYP9 family attained 62.3%. The transcription start site deduced by NNPP analysis sofeware was coincident with the result deduced according to the total length cDNA of CYP9A22. The combinatopm site of transcription factor analyzed by TFSEARCH 1.3 shows that the upriver sequence of transcription start site contains not only the nuclear structure sequence of promoter such as TATA-box and CAAT-box, but also several combination sites of transcription factors such as GATA-1,CdxA,Dfd, et al.
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