Cry1Ab13杀虫基因的PCR诱变及功能验证
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摘要
【目的】通过易错PCR技术克隆一种新的Cry1Ab13基因,以利用Cry类Bt基因提高作物抗虫性,丰富基因资源。【方法】利用易错PCR技术对苏云金芽孢杆菌Cry1Ab13基因进行随机诱变,构建突变体文库,筛选获得突变株Cry1Ab13-1,将该基因构建原核重组表达载体后转化到大肠杆菌中进行异源表达,并利用表达的目的蛋白进行抗虫鉴定。【结果】序列分析表明,突变株Cry1Ab13-1基因序列全长2 036bp,与原始碱基序列的一致性为97.79%,有2个碱基发生突变,导致该基因编码的氨基酸序列中有2个氨基酸改变,分别是第130位由脯氨酸变成丝氨酸,第383位由丝氨酸变成苏氨酸;改变后的氨基酸序列与原始氨基酸序列一致性为96.97%。在线分析表明,该基因与已知过敏源的序列同源性低于35%,不存在致敏性。SDS-PAGE电泳分析表明,表达蛋白分子质量大小为79.5ku,与预期结果相符。抗虫鉴定表明,处理72h时表达的目的蛋白对小菜蛾(Plutella xyllostella)幼虫的致死率达87.88%,明显高于未突变基因对照组的幼虫死亡率;对亚洲玉米螟(Ostrinia nubilalis)幼虫的致死率达81.11%,而且存活的亚洲玉米螟幼虫的生长发育受到明显抑制。【结论】成功克隆了Cry1Ab13-1抗虫基因,且该基因对小菜蛾幼虫和亚洲玉米螟幼虫均具有高效的杀虫活性。
【Objective】A novel Cry1 Ab13 gene was cloned successfully by error-prone PCR technology to enrich gene resources for improving crop insect resistance using the Cry-type Bt gene.【Method】The experiment conducted random mutagenesis on bacillus thuringiensis Cry1 Ab13 gene with error-prone PCR technology,built mutant library,and obtained mutant strain Cry1 Ab13-1,which was transformed into Escherichia coli for heterologous protein expression.The protein encoded with Cry1 Ab13-1 gene was used for identification of insect resistance.【Result】The sequence analysis showed that the length of mutant strain Cry1 Ab13-1 gene sequence was 2 036 bp,with the consistency of 97.79%to original base sequence.There were two bases mutates among the amino acid sequence,causing two amino acids changes at the130 th from proline to serine and the 383 th from serine to threonine.The consistency between the changed and original amino acid sequences was 96.97%.The on-line analysis showed that the homology between the gene and the known allergen sequence was less than 35% without sensitization.The SDS-PAGE electro-phoretic showed that the molecular weight of expressed protein was about 79.5 ku,which was coincident with expectation.Insect resistance identification showed that the larval fatality rate of expressed objective protein on Plutella xyllostella reached 87.88%,significantly higher than the non-mutant gene control group.The larval fatality rate of Asian Ostrinia nubilalis reached 81.11%,and the growth and development of survival Asian Ostrinia nubilalis larval were significantly inhibited.【Conclusion】This study successfully cloned Cry1 Ab13-1 insect gene and the gene had efficient insecticidal activity against Asian corn borer larvae and diamondback moth larvae.
【Objective】A novel Cry1 Ab13 gene was cloned successfully by error-prone PCR technology to enrich gene resources for improving crop insect resistance using the Cry-type Bt gene.【Method】The experiment conducted random mutagenesis on bacillus thuringiensis Cry1 Ab13 gene with error-prone PCR technology,built mutant library,and obtained mutant strain Cry1 Ab13-1,which was transformed into Escherichia coli for heterologous protein expression.The protein encoded with Cry1 Ab13-1 gene was used for identification of insect resistance.【Result】The sequence analysis showed that the length of mutant strain Cry1 Ab13-1 gene sequence was 2 036 bp,with the consistency of 97.79%to original base sequence.There were two bases mutates among the amino acid sequence,causing two amino acids changes at the130 th from proline to serine and the 383 th from serine to threonine.The consistency between the changed and original amino acid sequences was 96.97%.The on-line analysis showed that the homology between the gene and the known allergen sequence was less than 35% without sensitization.The SDS-PAGE electro-phoretic showed that the molecular weight of expressed protein was about 79.5 ku,which was coincident with expectation.Insect resistance identification showed that the larval fatality rate of expressed objective protein on Plutella xyllostella reached 87.88%,significantly higher than the non-mutant gene control group.The larval fatality rate of Asian Ostrinia nubilalis reached 81.11%,and the growth and development of survival Asian Ostrinia nubilalis larval were significantly inhibited.【Conclusion】This study successfully cloned Cry1 Ab13-1 insect gene and the gene had efficient insecticidal activity against Asian corn borer larvae and diamondback moth larvae.
引文
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[9]李怡萍,梁革梅,陈豪,等.苏云金芽孢杆菌杀虫机理及害虫对其抗性机制的研究进展[J].西北农林科技大学学报(自然科学版),2010,38(9):118-128.Li Y P,Liang G M,Chen H,et al.Progress in insecticidal mechanism of Bt and resistance mechanism of pest insect to Bt[J].Journal of Northwest A&F University(Nat Sci Ed),2010,38(9):118-128.
[10]Barton K A,Whiteley H R,Yang N S.Bacillus thuringiensis Delta-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidoptern insects[J].Plant Physiol,1987,85:1103-1109.
[11]郭三堆,王远,孙国清,等.中国转基因棉花研发应用二十年[J].中国农业科学,2015,48(17):3372-3387.Guo S D,Wang Y,Suo G Q,et al.Twenty years of research and application of transgenic cotton in China[J].Scientia Agricultura Sinica,2015,48(17):3372-3387.
[12]王阳,杨朔,尹悦佳,等.分子改造Cry蛋白基因的转基因玉米创制及其抗虫功能评价[J].玉米科学,2015,23(6):27-34.Wang Y,Yang S,Yin Y J,et al.Rational mutation of cry protein and the insect resistance assessment in the transgenic maize[J].Journal of Maize Sciences,2015,23(6):27-34.
[13]关雄,蔡俊.我国苏云金杆菌研究60年[J].微生物学通报,2014,41(3):459-465.Guan X,Cai J.Bacillus thuringiensis:sixty years of research development and commercial applications in China[J].Microbiology China,2014,41(3):459-465.
[14]高义平,赵和,吕梦雨,等.易错PCR研究进展及应用[J].核农学报,2013,27(5):607-612.Gao Y P,Zhao H,LüM Y,et al.Research progress and application of error-prone PCR[J].Journal of Nuclear Agricultural Sciences,2013,27(5):607-612.
[15]Leung D W,Chen E,Goeddel D V.A method for random mutagen of a defined DNA segment using a modified polymerase chain recation[J].Technique,1989,1:11-15.
[16]Cadwell C,Joyce G F.Randomization of genes by PCR mutagenesis[J].PCR Methods and Applications,1992,2:28-33.
[17]Shelly G,Arbind K,Jagdeep K.Disruption of N terminus long range non covalent interactions shifted temd.opt 25℃to cold:evolution of point mutant Bacillus lipase by error prone PCR[J].Gene,2015,10:29-31.
[18]郭兆将,康师,吴青君,等.小菜蛾饲养技术与规范[J].应用昆虫学报,2015,52(2):492-497.Guo Z J,Kang S,Wu Q J,et al.Technical specifications for the mass-rearing of the diamondback moth,Plutella xylostella(L.)[J].Chinese Journal of Applied Entomology,2015,52(2):492-497.
[19]袁志华,王文强,王振营,等.亚洲玉米螟的寄主植物种类[J].植物保护学报,2015,42(6):957-964.Yuan Z H,Wang W Q,Wang Z Y,et al.Host plants of the Asian corn borer,Ostrinia furnacalis(Guenée)(Lepidoptera:Crambidae)[J].Journal of Plant Protection,2015,42(6):957-964.
[20]秦秀林,钱江潮,储炬,等.优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率[J].生物技术通报,2016,10(6):211-217.Qin X L,Qian J C,Chu J,et al.High-error-rate random mutagenesis of GAP promoter in pichia pastoris using an optimited error prone PCR[J].Biotechnology Bulletin,2016,10(6):211-217.
[21]曾家豫,郑群,廖世奇,等.大豆过氧化物酶基因随机突变文库的构建[J].大豆科学,2011,30(5):731-737.Zeng J Y,Zheng Q,Liao S Q,et al.Construction of soybean peroxidase gene random mutation library[J].Soybean Science,2011,30(5):731-737.
[22]刘卫晓,钱世君.酶分子体外定向进化的研究方法[J].微生物学报,2004,31(2):100-104.Liu W X,Qian S J.Methods of the directed molecular evolution of enzyme in vitro[J].Microbiology China,2004,31(2):100-104.
[23]宋萍,南宫自艳,张杰,等.苏云金芽胞杆菌Cry1Ia32基因克隆和原核表达的研究[J].河北农业大学学报,2015,38(5):48-52.Song P,Nangong Z Y,Zhang J,et al.Cloning and expression of Cry1Ia32gene frome Bacilius thuringiensis isolate[J].Journal of Agricultural University of Hebei,2015,38(5):48-52.
[24]乔利,郑坚武,成卫宁,等.不同饲料配方对亚洲玉米螟生长发育和繁殖的影响[J].西北农林科技大学学报(自然科学版),2008,36(5):109-112.Qiao L,Zheng J W,Cheng W N,et al.Impact of 4different artificial fodders on life span of asian corn borer,Ostrinia furnacalis(Guenee)[J].Journal of Northwest A&F University(Nat Sci Ed),2008,36(5):109-112.
[25]岳润清,铁双贵,陈小洁,等.人工改造基因Cry1Abm的合成、原核表达及其抗虫鉴定[J].玉米科学,2013,21(6):21-25.Yue R Q,Tie S G,Chen X J,et al.Synthesis of the modified Cry1Abm gene,expressionin E.coli and bioassay[J].Journal of Maize Sciences,2013,21(6):21-25.
[26]任羽,刘华梅,宋福平,等.苏云金芽孢杆菌Cry1Ca7蛋白定点突变对甜菜夜蛾杀虫活性的影响[J].微生物学报,2008,48(6):733-738.Ren Y,Liu H M,Song F P,et al.Effect of Cry1Ca7protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hübne[J].Acta Microbiologica Sinica,2008,48(6):733-738.
[27]Drummond D A,Iverson B L,Georgiou G,et al.Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins[J].Journal of Molecular Biology,2005,350(4):806-816.
[28]黄瑛,蔡勇,杨江科,等.基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化[J].生物工程学报,2008,24(3):445-451.Huang Y,Cai Y,Yang J K,et al.Directed evolution of lipase of Bacillus pumilus YZ02by error-prone PCR[J].Chinese Journal of Biotechnology,2008,24(3):445-451.
[29]余宗兰,贺利业.苏云金芽胞杆菌cry1Ie新基因的克隆、表达与生物活性测定[J].植物保护学报,2016,43(2):201-206.Yu Z L,He L Y.Cloning,expression,and biological activity of a new cry1Ie gene from Bacillus thuringiensis strain[J].Journal of Plant Protection,2016,43(2):201-206.
[2]陈晓明,王程龙,博瑞.中国农药使用现状及对策建议[J].农药科学与管理,2016,37(2):4-8.Chen X M,Wang C L,Bo R.Current situation of Chinese pesticide application and policy suggestions[J].Pesticide Science and Administration,2016,37(2):4-8.
[3]Breed.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.Breed.Bergey’s manual of determinative bacteriology[M].Beijing:Science Press,1984.
[4]郭三堆.植物Bt基因工程研究进展[J].中国农业科学,1995,28(5):8-13.Guo S D.Engineering of insect-resistant plant with Bbacillus thuringiensis crystal protein genes[J].Scientia Agricultura Sinica,1995,28(5):8-13.
[5]叶恭银,陈洋,田俊策,等.转Bt基因抗虫作物对非靶标生物的影响[J].植物保护,2011,37(6):1-10.Ye G Y,Chen Y,Tian J C,et al.The effects of transgenic Bt insect-resistant crops on non-target organisma[J].Plant Protection,2011,37(6):1-10.
[6]杨艳,李云河,曹凤勤,等.转Bt基因抗虫作物对鳞翅目非靶标昆虫生态影响的研究进展[J].生物安全学报,2014,23(4):224-237.Yang Y,Li Y H,Cao F Q,et al.Progress in the assessment of ecological effects of insect-resistant Bt crops on non-target of lepidopteran insects[J].Journal of Biosafety,2014,23(4):224-237.
[7]Ernest S.A relational database giving known insecticidal activities of the delta-endotoxins[J/OL].(2016-11-20).http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.
[8]林珠凤,陈海燕,吉训聪,等.害虫对转Bt基因作物的抗性及治理[J].热带生物学报,2015,6(4):497-503.Lin Z F,Chen H Y,Ji X C,et al.Insect resistance to Bt crops:possible mechanism and resistance management strategy[J].Journal of Tropical Biology,2015,6(4):497-503.
[9]李怡萍,梁革梅,陈豪,等.苏云金芽孢杆菌杀虫机理及害虫对其抗性机制的研究进展[J].西北农林科技大学学报(自然科学版),2010,38(9):118-128.Li Y P,Liang G M,Chen H,et al.Progress in insecticidal mechanism of Bt and resistance mechanism of pest insect to Bt[J].Journal of Northwest A&F University(Nat Sci Ed),2010,38(9):118-128.
[10]Barton K A,Whiteley H R,Yang N S.Bacillus thuringiensis Delta-endotoxin expressed in transgenic Nicotiana tabacum provides resistance to lepidoptern insects[J].Plant Physiol,1987,85:1103-1109.
[11]郭三堆,王远,孙国清,等.中国转基因棉花研发应用二十年[J].中国农业科学,2015,48(17):3372-3387.Guo S D,Wang Y,Suo G Q,et al.Twenty years of research and application of transgenic cotton in China[J].Scientia Agricultura Sinica,2015,48(17):3372-3387.
[12]王阳,杨朔,尹悦佳,等.分子改造Cry蛋白基因的转基因玉米创制及其抗虫功能评价[J].玉米科学,2015,23(6):27-34.Wang Y,Yang S,Yin Y J,et al.Rational mutation of cry protein and the insect resistance assessment in the transgenic maize[J].Journal of Maize Sciences,2015,23(6):27-34.
[13]关雄,蔡俊.我国苏云金杆菌研究60年[J].微生物学通报,2014,41(3):459-465.Guan X,Cai J.Bacillus thuringiensis:sixty years of research development and commercial applications in China[J].Microbiology China,2014,41(3):459-465.
[14]高义平,赵和,吕梦雨,等.易错PCR研究进展及应用[J].核农学报,2013,27(5):607-612.Gao Y P,Zhao H,LüM Y,et al.Research progress and application of error-prone PCR[J].Journal of Nuclear Agricultural Sciences,2013,27(5):607-612.
[15]Leung D W,Chen E,Goeddel D V.A method for random mutagen of a defined DNA segment using a modified polymerase chain recation[J].Technique,1989,1:11-15.
[16]Cadwell C,Joyce G F.Randomization of genes by PCR mutagenesis[J].PCR Methods and Applications,1992,2:28-33.
[17]Shelly G,Arbind K,Jagdeep K.Disruption of N terminus long range non covalent interactions shifted temd.opt 25℃to cold:evolution of point mutant Bacillus lipase by error prone PCR[J].Gene,2015,10:29-31.
[18]郭兆将,康师,吴青君,等.小菜蛾饲养技术与规范[J].应用昆虫学报,2015,52(2):492-497.Guo Z J,Kang S,Wu Q J,et al.Technical specifications for the mass-rearing of the diamondback moth,Plutella xylostella(L.)[J].Chinese Journal of Applied Entomology,2015,52(2):492-497.
[19]袁志华,王文强,王振营,等.亚洲玉米螟的寄主植物种类[J].植物保护学报,2015,42(6):957-964.Yuan Z H,Wang W Q,Wang Z Y,et al.Host plants of the Asian corn borer,Ostrinia furnacalis(Guenée)(Lepidoptera:Crambidae)[J].Journal of Plant Protection,2015,42(6):957-964.
[20]秦秀林,钱江潮,储炬,等.优化易错PCR条件以提高毕赤酵母GAP启动子文库突变效率[J].生物技术通报,2016,10(6):211-217.Qin X L,Qian J C,Chu J,et al.High-error-rate random mutagenesis of GAP promoter in pichia pastoris using an optimited error prone PCR[J].Biotechnology Bulletin,2016,10(6):211-217.
[21]曾家豫,郑群,廖世奇,等.大豆过氧化物酶基因随机突变文库的构建[J].大豆科学,2011,30(5):731-737.Zeng J Y,Zheng Q,Liao S Q,et al.Construction of soybean peroxidase gene random mutation library[J].Soybean Science,2011,30(5):731-737.
[22]刘卫晓,钱世君.酶分子体外定向进化的研究方法[J].微生物学报,2004,31(2):100-104.Liu W X,Qian S J.Methods of the directed molecular evolution of enzyme in vitro[J].Microbiology China,2004,31(2):100-104.
[23]宋萍,南宫自艳,张杰,等.苏云金芽胞杆菌Cry1Ia32基因克隆和原核表达的研究[J].河北农业大学学报,2015,38(5):48-52.Song P,Nangong Z Y,Zhang J,et al.Cloning and expression of Cry1Ia32gene frome Bacilius thuringiensis isolate[J].Journal of Agricultural University of Hebei,2015,38(5):48-52.
[24]乔利,郑坚武,成卫宁,等.不同饲料配方对亚洲玉米螟生长发育和繁殖的影响[J].西北农林科技大学学报(自然科学版),2008,36(5):109-112.Qiao L,Zheng J W,Cheng W N,et al.Impact of 4different artificial fodders on life span of asian corn borer,Ostrinia furnacalis(Guenee)[J].Journal of Northwest A&F University(Nat Sci Ed),2008,36(5):109-112.
[25]岳润清,铁双贵,陈小洁,等.人工改造基因Cry1Abm的合成、原核表达及其抗虫鉴定[J].玉米科学,2013,21(6):21-25.Yue R Q,Tie S G,Chen X J,et al.Synthesis of the modified Cry1Abm gene,expressionin E.coli and bioassay[J].Journal of Maize Sciences,2013,21(6):21-25.
[26]任羽,刘华梅,宋福平,等.苏云金芽孢杆菌Cry1Ca7蛋白定点突变对甜菜夜蛾杀虫活性的影响[J].微生物学报,2008,48(6):733-738.Ren Y,Liu H M,Song F P,et al.Effect of Cry1Ca7protein modified by site-directed mutagenesis on inhibiting Spodoptera exigua Hübne[J].Acta Microbiologica Sinica,2008,48(6):733-738.
[27]Drummond D A,Iverson B L,Georgiou G,et al.Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins[J].Journal of Molecular Biology,2005,350(4):806-816.
[28]黄瑛,蔡勇,杨江科,等.基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化[J].生物工程学报,2008,24(3):445-451.Huang Y,Cai Y,Yang J K,et al.Directed evolution of lipase of Bacillus pumilus YZ02by error-prone PCR[J].Chinese Journal of Biotechnology,2008,24(3):445-451.
[29]余宗兰,贺利业.苏云金芽胞杆菌cry1Ie新基因的克隆、表达与生物活性测定[J].植物保护学报,2016,43(2):201-206.Yu Z L,He L Y.Cloning,expression,and biological activity of a new cry1Ie gene from Bacillus thuringiensis strain[J].Journal of Plant Protection,2016,43(2):201-206.