桔小实蝇钠离子通道基因和P450基因RNAi载体构建及转化
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摘要
桔小实蝇是重要的柑橘害虫,目前已对多种杀虫剂产生不同程度的抗性,化学防治方法往往效果不理想,近年来植物转基因抗虫育种技术表现出巨大的潜力,这为更好地防治桔小实蝇提供了新思路。本研究为获得桔小实蝇抗性转基因柑橘,对桔小实蝇细胞色素P450基因(cytochrome P450 monoxygenases, P450)和电压门控钠离子通道基因(sodium channel, SC)进行核苷酸序列比对,分别克隆得到片段大小为590 bp和550 bp的P450基因和SC基因的正反目标片段,利用植物表达载体pFGC5941,将基因正反向片段与Intron两端连接,成功构建RNA干扰(RNAi)载体"p FGC-P1F1R1/P2F2"和"pFGC-C1R1/C2R2"。本研究利用XhoⅠ-NcoⅠ、XbaⅠ-Bam HⅠ酶切位点对质粒和载体进行双酶切分析实验,克隆测序得到大小为590 bp和550 bp的两个酶切片段,经核苷酸序列比对分析,验证其与插入片段一致,说明RNAi载体构建成功。通过农杆菌遗传转化体系,将外源重组载体整合到柑橘基因组中,经除草剂筛选、PCR检测分别得到12株转P450基因阳性苗和9株转钠离子通道基因阳性苗,这为后期柑橘抗桔小实蝇效果评价提供了实验材料。
Bactrocera dorsalis is an important citrus pest. It has already produced various degrees of resistance to various insecticides. The effect of chemical control methods is not satisfactory. In recent years, transgenic insect-resistant breeding techniques have shown great potential, which provides novel insights for the better control of Bactrocera dorsalis. In this study, the nucleotide sequences of cytochrome P450(cytochrome P450monoxygenases) and voltage-gated sodium channel(sodium channel, SC) genes were compared to obtain transgenic citrus fruit resistant to B. dorsalis. The forward and reverse target fragments of P450 gene and SC gene with the size of 590 bp and 550 bp were cloned, respectively. Using the plant expression vector pFGC5941, the gene forward and reverse fragments were ligated with both ends of Intron to successfully construct an RNA interference(RNAi) vector, "pFGC-P1F1R1/P2F2" and "pFGC-C1R1/C2R2". In this study, the restriction enzyme digestion sites of XhoⅠ-NcoⅠ and XbaⅠ-Bam HⅠ were used to carry out double enzyme digestion analysis of plasmids and vectors. By cloning and sequencing, two fragments of 590 bp and 550 bp were obtained, which was consistent with the insert fragments based on nucleotide sequence analysis, indicating the construction of the RNAi vector was successful. Through the Agrobacterium genetic transformation system, the exogenous recombinant vector was integrated into the citrus genome. Twelve transgenic plants with positive P450 gene and nine transgenic plants with positive sodium ion channels were obtained by herbicide screening and PCR detection, respectively. This study would provide experimental materials for evaluation of the resistant effect of the citrus fruit fly Bactrocera dorsalis.
Bactrocera dorsalis is an important citrus pest. It has already produced various degrees of resistance to various insecticides. The effect of chemical control methods is not satisfactory. In recent years, transgenic insect-resistant breeding techniques have shown great potential, which provides novel insights for the better control of Bactrocera dorsalis. In this study, the nucleotide sequences of cytochrome P450(cytochrome P450monoxygenases) and voltage-gated sodium channel(sodium channel, SC) genes were compared to obtain transgenic citrus fruit resistant to B. dorsalis. The forward and reverse target fragments of P450 gene and SC gene with the size of 590 bp and 550 bp were cloned, respectively. Using the plant expression vector pFGC5941, the gene forward and reverse fragments were ligated with both ends of Intron to successfully construct an RNA interference(RNAi) vector, "pFGC-P1F1R1/P2F2" and "pFGC-C1R1/C2R2". In this study, the restriction enzyme digestion sites of XhoⅠ-NcoⅠ and XbaⅠ-Bam HⅠ were used to carry out double enzyme digestion analysis of plasmids and vectors. By cloning and sequencing, two fragments of 590 bp and 550 bp were obtained, which was consistent with the insert fragments based on nucleotide sequence analysis, indicating the construction of the RNAi vector was successful. Through the Agrobacterium genetic transformation system, the exogenous recombinant vector was integrated into the citrus genome. Twelve transgenic plants with positive P450 gene and nine transgenic plants with positive sodium ion channels were obtained by herbicide screening and PCR detection, respectively. This study would provide experimental materials for evaluation of the resistant effect of the citrus fruit fly Bactrocera dorsalis.
引文
Baum J.A.,Bogaert T.,Clinton W.,Heck G.R.,Feldmann P.,Ilagan O.,Johnson S.,Plaetinck G.,Munyikwa T.,Pleau M.,Vaughn T.,and Roberts J.,2007,Control of coleopteran insect pests through RNA interference,Nat.Biotechnol.,25(11):1322-1326
Bautista M.A.M.,Miyata T.,M iura K.,and Tanaka T.,2009,RNA interference mediated knockdown of a cytochrome P450,CYP6BG1,from the diamondback moth,Plutellaxylostella,reduces larval resistance to permethrin,Insect Biochem.Mol.Biol.,39(1):38-46
Boykin L.M.,Schutze M.K.,Krosch M.N.,Chomic A.,Chapman T.A.,Englezou A.,Armstrong K.F.,Clarke A.R.,Hailstones D.,and Cameron S.L.,2014,Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex(Diptera:Tephritidae)does not support current taxonomy,J.Appl.Entomol.,138(4):235-253
Deka S.,and Barthakur S.,2010,Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas(Lepidoptera:Crambidae)resistance in rice,Biotechnol.Adv.,28(1):70-81
Fabrick J.A.,Mathew L.G.,Tabashnik B.E.,and Li X.C.,2011,Insertion of an intact CR1 retrotransposon in a cadherin gene linked with bt resistance in the pink bollworm,Pectinophora gossypiella,Insect Mol.Biol.,20(5):651-665
Fire A.,Xu S.,Montgomery M.K.,Kostas S.A.,Driver S.E.,and Mello C.C.,1998,Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans,Nature,391(6669):806-811
Hidaka T.,Omura M.,Ugaki M.,Tomiyama M.,Kato A.,and Ohshima M.,1990,Agrobacterium-mediated transformation and regeneration of Citrus spp.from suspension cells,Japanese Journal of Breeding,40(2):199-207
Hsu J.C.,and Feng H.T.,2000,Insecticide susceptibility of the oriental fruit fly(Bactrocera dorsalis(Hendel))(Diptera:Tephritidae)in Taiwan,Chinese Journal of Entomology,20:109-118
Jin S.,Singh N.D.,Li L.,Zhang X.,and Daniell H.,2015,Engineered chloroplast dsRNA silences cytochrome P450monooxygenase,V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation,Plant Biotechnol.J.,13(3):435-446
Koziel M.G.,Beland G.L.,Bowman C.,Carozzi N.B.,Crenshaw R.,Crossland L.,Dawson J.,Desai N.,Hill M.,Kadwell S.,Launis K.,Lewis K.,Maddox D.,McPherson K.,Meghji M.R.,Merlin E.,Rhodes R.,Warren G.W.,Wright M.,and Evola S.V.,1993,Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis,Nat.Biotechnol.,11(2):194-200
Kumar P.,Pandit S.S.,and Baldwin I.T.,2012,Tobacco rattle virus vector:a rapid and transient means of silencing manducasexta genes by plant mediated RNA interference,PLoSOne,7(2):e31347
Kupferschmidt K.,2013,A lethal dose of RNA,Science,341(6147):732-733
Lin J.T.,Zeng L.,Liang G.W.,Lu Y.Y.,and Wang L.,2005,Distribution spatial pattern of Bactrocera(Bactrocera)dorsalis males,Huanan Nongye Daxue Xuebao(Journal of South China Agricultural University),26(2):43-46(林进添,曾玲,梁广文,陆永跃,王琳,2005,桔小实蝇雄成虫的空间分布格局,华南农业大学学报,26(2):43-46)
Mao Y.B.,Cai W.J.,Wang J.W.,Hong G.J.,Tao X.Y.,Wang L.J.,Huang Y.P.,and Chen X.Y.,2007,Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impair slarval tolerance of gossypol,Nat.Biotechnol.,25(11):1307-1313
Moore G.A.,Jacono C.C.,Neidigh J.L.,Lawrence S.D.,and Cline K.,1992,Agrobacterium-mediated transformation of citrus,stem segments and regeneration of transgenic plants,Plant Cell Rep.,11(5-6):238-242
Nandety R.S.,Kuo Y.W.,Nouri S.,and Falk B.W.,2015,Emerging strategies for RNA interference(RNAi)applications in insects,Bioengineered Bugs,6(1):8-19
Palli S.R.,2014,RNA interference in Colorado potato beetle:steps toward development of dsRNA as a commercial insecticide,Curr.Opin.Insect.Sci.,6:1-8
Saurabh S.,Vidyarthi A.S.,and Prasad D.,2014,RNA interference:concept to reality in crop improvement,Planta,239(3):543-564
Tao X.Y.,Xue Y.I.,Huang Y.P.,Chen X.Y.,and Mao Y.B.,2012,Gossypol-enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide,Mol.Ecol.,21(17):4371-4385
Wang Y.Q.,Li Y.G.,Wang T.,Zhang Y.H.,Sun M.Y.,and Li W.B.,2015,Cloning of gene GmAKT1 from soybean and construction of its sense and antisense plant expression vector,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):143-148(王英琪,李永光,王涛,张宇航,孙铭阳,李文滨,2015,大豆GmAKT1基因的克隆及其正反义植物表达载体构建,基因组学与应用生物学,34(1):143-148)
Yu X.D.,Liu Z.C.,Huang S.L.,Chen Z.Q.,Sun,Y.W.,Duan P.F.,Ma Y.Z.,and Xia L.Q.,2016,RNAi-mediated plant protection against aphids,Pest Manag.Sci.,72(6):1090-1098
Zhang Y.P.,and Li D.S.,2007,Advances in biological control research of Bactrocera dorsalis(Hendel),Kunchong Tiandi(Natural Enemies of Insects),29(4):173-181(章玉苹,李敦松,2007,桔小实蝇生物防治研究进展,昆虫天敌,29(4):173-181)
Zheng C.Y.,Zeng L.,and Xu Y.J.,2016,Effect of sweeteners on the survival andbehavior of Bactrocera dorsalis(Hendel)(Diptera:Tephritidae),Pest Manag.Sci.,72(5):990-960
Zhu J.Q.,Liu S.,Ma Y.,Zhang J.Q.,Qi H.S.,Wei Z.J.,Yao Q.,Zhang W.Q.,and Li S.,2012,Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR,PLoS One,7(6):e38572
Zottiand M.J.,and Smagghe G.,2015,RNAi technology for insect management and protection of beneficial insects from diseases:lessons,challenges and risk assessments,Neotrop.Entomol.,44(3):197-213
Bautista M.A.M.,Miyata T.,M iura K.,and Tanaka T.,2009,RNA interference mediated knockdown of a cytochrome P450,CYP6BG1,from the diamondback moth,Plutellaxylostella,reduces larval resistance to permethrin,Insect Biochem.Mol.Biol.,39(1):38-46
Boykin L.M.,Schutze M.K.,Krosch M.N.,Chomic A.,Chapman T.A.,Englezou A.,Armstrong K.F.,Clarke A.R.,Hailstones D.,and Cameron S.L.,2014,Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex(Diptera:Tephritidae)does not support current taxonomy,J.Appl.Entomol.,138(4):235-253
Deka S.,and Barthakur S.,2010,Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas(Lepidoptera:Crambidae)resistance in rice,Biotechnol.Adv.,28(1):70-81
Fabrick J.A.,Mathew L.G.,Tabashnik B.E.,and Li X.C.,2011,Insertion of an intact CR1 retrotransposon in a cadherin gene linked with bt resistance in the pink bollworm,Pectinophora gossypiella,Insect Mol.Biol.,20(5):651-665
Fire A.,Xu S.,Montgomery M.K.,Kostas S.A.,Driver S.E.,and Mello C.C.,1998,Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans,Nature,391(6669):806-811
Hidaka T.,Omura M.,Ugaki M.,Tomiyama M.,Kato A.,and Ohshima M.,1990,Agrobacterium-mediated transformation and regeneration of Citrus spp.from suspension cells,Japanese Journal of Breeding,40(2):199-207
Hsu J.C.,and Feng H.T.,2000,Insecticide susceptibility of the oriental fruit fly(Bactrocera dorsalis(Hendel))(Diptera:Tephritidae)in Taiwan,Chinese Journal of Entomology,20:109-118
Jin S.,Singh N.D.,Li L.,Zhang X.,and Daniell H.,2015,Engineered chloroplast dsRNA silences cytochrome P450monooxygenase,V-ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa zea larval development and pupation,Plant Biotechnol.J.,13(3):435-446
Koziel M.G.,Beland G.L.,Bowman C.,Carozzi N.B.,Crenshaw R.,Crossland L.,Dawson J.,Desai N.,Hill M.,Kadwell S.,Launis K.,Lewis K.,Maddox D.,McPherson K.,Meghji M.R.,Merlin E.,Rhodes R.,Warren G.W.,Wright M.,and Evola S.V.,1993,Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis,Nat.Biotechnol.,11(2):194-200
Kumar P.,Pandit S.S.,and Baldwin I.T.,2012,Tobacco rattle virus vector:a rapid and transient means of silencing manducasexta genes by plant mediated RNA interference,PLoSOne,7(2):e31347
Kupferschmidt K.,2013,A lethal dose of RNA,Science,341(6147):732-733
Lin J.T.,Zeng L.,Liang G.W.,Lu Y.Y.,and Wang L.,2005,Distribution spatial pattern of Bactrocera(Bactrocera)dorsalis males,Huanan Nongye Daxue Xuebao(Journal of South China Agricultural University),26(2):43-46(林进添,曾玲,梁广文,陆永跃,王琳,2005,桔小实蝇雄成虫的空间分布格局,华南农业大学学报,26(2):43-46)
Mao Y.B.,Cai W.J.,Wang J.W.,Hong G.J.,Tao X.Y.,Wang L.J.,Huang Y.P.,and Chen X.Y.,2007,Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impair slarval tolerance of gossypol,Nat.Biotechnol.,25(11):1307-1313
Moore G.A.,Jacono C.C.,Neidigh J.L.,Lawrence S.D.,and Cline K.,1992,Agrobacterium-mediated transformation of citrus,stem segments and regeneration of transgenic plants,Plant Cell Rep.,11(5-6):238-242
Nandety R.S.,Kuo Y.W.,Nouri S.,and Falk B.W.,2015,Emerging strategies for RNA interference(RNAi)applications in insects,Bioengineered Bugs,6(1):8-19
Palli S.R.,2014,RNA interference in Colorado potato beetle:steps toward development of dsRNA as a commercial insecticide,Curr.Opin.Insect.Sci.,6:1-8
Saurabh S.,Vidyarthi A.S.,and Prasad D.,2014,RNA interference:concept to reality in crop improvement,Planta,239(3):543-564
Tao X.Y.,Xue Y.I.,Huang Y.P.,Chen X.Y.,and Mao Y.B.,2012,Gossypol-enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide,Mol.Ecol.,21(17):4371-4385
Wang Y.Q.,Li Y.G.,Wang T.,Zhang Y.H.,Sun M.Y.,and Li W.B.,2015,Cloning of gene GmAKT1 from soybean and construction of its sense and antisense plant expression vector,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):143-148(王英琪,李永光,王涛,张宇航,孙铭阳,李文滨,2015,大豆GmAKT1基因的克隆及其正反义植物表达载体构建,基因组学与应用生物学,34(1):143-148)
Yu X.D.,Liu Z.C.,Huang S.L.,Chen Z.Q.,Sun,Y.W.,Duan P.F.,Ma Y.Z.,and Xia L.Q.,2016,RNAi-mediated plant protection against aphids,Pest Manag.Sci.,72(6):1090-1098
Zhang Y.P.,and Li D.S.,2007,Advances in biological control research of Bactrocera dorsalis(Hendel),Kunchong Tiandi(Natural Enemies of Insects),29(4):173-181(章玉苹,李敦松,2007,桔小实蝇生物防治研究进展,昆虫天敌,29(4):173-181)
Zheng C.Y.,Zeng L.,and Xu Y.J.,2016,Effect of sweeteners on the survival andbehavior of Bactrocera dorsalis(Hendel)(Diptera:Tephritidae),Pest Manag.Sci.,72(5):990-960
Zhu J.Q.,Liu S.,Ma Y.,Zhang J.Q.,Qi H.S.,Wei Z.J.,Yao Q.,Zhang W.Q.,and Li S.,2012,Improvement of pest resistance in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR,PLoS One,7(6):e38572
Zottiand M.J.,and Smagghe G.,2015,RNAi technology for insect management and protection of beneficial insects from diseases:lessons,challenges and risk assessments,Neotrop.Entomol.,44(3):197-213