Cry1Ia蛋白的表达与纯化
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摘要
苏云金芽孢杆菌表达的Cry1Ia蛋白对鳞翅目和鞘翅目昆虫具有较好的杀虫活性。为便于后期评价不同的Cry1Ia蛋白,建立了Cry1Ia蛋白的表达与纯化体系。将一个cry1Ia基因的编码基因接入pET28aDel表达载体,并将其转化大肠杆菌BL21(DE3)star菌株。在大肠杆菌中诱导Cry1Ia蛋白的表达,并与Bt中的表达产物进行比较。最后,利用Cry1Ia蛋白C端包含的组氨酸标签,对大肠杆菌表达的Cry1Ia蛋白进行纯化。结果显示,大肠杆菌中Cry1Ia蛋白随着表达时间的延长,产物积累显著,并且其表达量高于Bt菌株,因此,其更适宜生产完整的Cry1Ia蛋白。对大肠杆菌表达的Cry1Ia进行纯化,获得了理想的结果。研究构建了Cry1Ia蛋白的大肠杆菌表达体系,并成功对其进行纯化,可为进一步研究此类蛋白的杀虫活性和机制奠定基础。
Cry1 Ia proteins of Bacillus thuringiensis showed ideal insecticidal activity against Lepidoptera and Coleoptera pests. To evaluate different Cry1 Ia proteins, the expression and purification system of Cry1 Ia protein was established. A cry1 Ia gene was inserted into the pET28 aDel expression vector and transformed into Escherichia coli BL21(DE3)star strain. The expression of Cry1 Ia protein was induced and were compared with that of Bt strain. Finally, the Esoherichia coli expressed Cry1 Ia protein was purified by the histidine tag located at the C-terminus. The results showed that the Cry1 Ia protein in Escherichia coli accumulated significantly with the prolongation of expression time, and the expression level was higher than that of Bt strain, so it was more suitable to produce the intact Cry1 Ia protein.The constructed Cry1 Ia protein could be purified successfully. In this study, the Escherichia coli expression and purification system of Cry1 Ia protein was established which laid a foundation for further study of the insecticidal activity and mechanism of these proteins.
Cry1 Ia proteins of Bacillus thuringiensis showed ideal insecticidal activity against Lepidoptera and Coleoptera pests. To evaluate different Cry1 Ia proteins, the expression and purification system of Cry1 Ia protein was established. A cry1 Ia gene was inserted into the pET28 aDel expression vector and transformed into Escherichia coli BL21(DE3)star strain. The expression of Cry1 Ia protein was induced and were compared with that of Bt strain. Finally, the Esoherichia coli expressed Cry1 Ia protein was purified by the histidine tag located at the C-terminus. The results showed that the Cry1 Ia protein in Escherichia coli accumulated significantly with the prolongation of expression time, and the expression level was higher than that of Bt strain, so it was more suitable to produce the intact Cry1 Ia protein.The constructed Cry1 Ia protein could be purified successfully. In this study, the Escherichia coli expression and purification system of Cry1 Ia protein was established which laid a foundation for further study of the insecticidal activity and mechanism of these proteins.
引文
[1] SAYYED A H,RAY MOND B,IBIZA-PALACIOS M S,et al. Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth,Plutella xylostella[J]. Appl Environ Microbiol,2004,70(12):7010-7017.
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[5] DONOVAN W P,ENGLMAN J T,DONOVAN J C,et al. Discovery and characterization of Sip1A:A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae[J]. Appl Microbiol Biotechnol,2006,72(4):713-719.
[6]ADALAT R,SALEEM F,CRICKMORE N,et al. In vivo crystallization of three-domain Cry toxins[J]. Toxins,2017,9(3):80-93.
[7] SCHNEPF E,CRICKMORE N,RIE J,et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol Mol Biol Rev,1998,62(3):775-806.
[8] BOHOROVA N,FRUTOS R,ROYER M,et al. Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize[J]. Theor Appl Genet,2001,103(6):817-826.
[9]彭琦,周子珊,张杰.苏云金芽孢杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[10] JURAT-FUENTES J L,CRICKMORE N. Specificity determinants for Cry insecticidal proteins:Insights from their mode of action[J]. J Invertebr Pathol,2017,142(S):5-10.
[11] KOSTICHKA K,WARREN G W,MULLINS M,et al. Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis:the cryV-encoded protein is expressed early in stationary phase[J].Journal of Bacteriology,1996,178(7):2141-2144.
[12] SOBER魷N M,PARDO-L魷PEZ L,L魷PEZ I,et al. Engineering modified Bt toxins to counter insect resistance[J]. Science,2007,318:1640-1642.
[13]张春鸽,赵灿,束长龙,等.不同启动子表达Cry1Ie蛋白的特性分析[J].生物技术通报,2012(11):192-196.
[14] LIAN Y,JIA Z,HE K,et al. Transgenic tobacco plants expressing synthetic cry1Ac and cry1Ie genes are more toxic to cotton bollworm than those containing one gene[J]. Chinese Science Bulletin,2008,53(9):1381-1387.
[15]LIU Y J,SONG F P,HE K L. Expression of a modified cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer[J]. Acta Biochim Biophys Sin(Shanghai),2004,36(4):309-313.
[16]杨召军,郎志宏,张杰,等.转Bt cry1Ah/cry1Ie双价基因抗虫玉米的研究[J].中国农业科技导报,2012,14(4):39-45.
[17]GAO J,ZHANG Y E. Transgenic rice expressing a fusion protein of Cry1Ab and Cry9Aa confers resistance to a broad spectrum of lepidopteran pests[J]. Crop Science,2011,51(6):2535-2543.
[18]高建华,王思宇,黄婷婷,等. Cry1Ia和Cry2Ab杂交分子的构建和表达[J].山西农业科学,2015,43(5):521-523,531.
[2] SCHNEPF E,CRICKMORE N,RIE J V,et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol Mol Biol Rev,1998,62(3):775-806.
[3] ARONSON A I,BECKMAN W,DUNN P. Bacillus thuringiensis and related insect pathogens[J]. Microbiol Rev,1986,50(1):1-24.
[4] ESTRUCH J J,WARREN G W,MULLINS M A,et al. Vip3A,a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects[J]. Proc Natl Acad Sci USA,1996,93(11):5389-5394.
[5] DONOVAN W P,ENGLMAN J T,DONOVAN J C,et al. Discovery and characterization of Sip1A:A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae[J]. Appl Microbiol Biotechnol,2006,72(4):713-719.
[6]ADALAT R,SALEEM F,CRICKMORE N,et al. In vivo crystallization of three-domain Cry toxins[J]. Toxins,2017,9(3):80-93.
[7] SCHNEPF E,CRICKMORE N,RIE J,et al. Bacillus thuringiensis and its pesticidal crystal proteins[J]. Microbiol Mol Biol Rev,1998,62(3):775-806.
[8] BOHOROVA N,FRUTOS R,ROYER M,et al. Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize[J]. Theor Appl Genet,2001,103(6):817-826.
[9]彭琦,周子珊,张杰.苏云金芽孢杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[10] JURAT-FUENTES J L,CRICKMORE N. Specificity determinants for Cry insecticidal proteins:Insights from their mode of action[J]. J Invertebr Pathol,2017,142(S):5-10.
[11] KOSTICHKA K,WARREN G W,MULLINS M,et al. Cloning of a cryV-type insecticidal protein gene from Bacillus thuringiensis:the cryV-encoded protein is expressed early in stationary phase[J].Journal of Bacteriology,1996,178(7):2141-2144.
[12] SOBER魷N M,PARDO-L魷PEZ L,L魷PEZ I,et al. Engineering modified Bt toxins to counter insect resistance[J]. Science,2007,318:1640-1642.
[13]张春鸽,赵灿,束长龙,等.不同启动子表达Cry1Ie蛋白的特性分析[J].生物技术通报,2012(11):192-196.
[14] LIAN Y,JIA Z,HE K,et al. Transgenic tobacco plants expressing synthetic cry1Ac and cry1Ie genes are more toxic to cotton bollworm than those containing one gene[J]. Chinese Science Bulletin,2008,53(9):1381-1387.
[15]LIU Y J,SONG F P,HE K L. Expression of a modified cry1Ie gene in E. coli and in transgenic tobacco confers resistance to corn borer[J]. Acta Biochim Biophys Sin(Shanghai),2004,36(4):309-313.
[16]杨召军,郎志宏,张杰,等.转Bt cry1Ah/cry1Ie双价基因抗虫玉米的研究[J].中国农业科技导报,2012,14(4):39-45.
[17]GAO J,ZHANG Y E. Transgenic rice expressing a fusion protein of Cry1Ab and Cry9Aa confers resistance to a broad spectrum of lepidopteran pests[J]. Crop Science,2011,51(6):2535-2543.
[18]高建华,王思宇,黄婷婷,等. Cry1Ia和Cry2Ab杂交分子的构建和表达[J].山西农业科学,2015,43(5):521-523,531.
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