苏云金芽胞杆菌新型cry1基因克隆、表达及活性分析
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一种世界上应用广泛的生防微生物,它具有高度的杀虫特异性和对人畜无害的优点。主要的杀虫成份是杀虫晶体蛋白,由cry和cyt基因编码。其中cry1类基因的表达产物对多种鳞翅目害虫具有较高的杀虫活性,但是由于检测方法的局限性,快速、准确地鉴定已知和未知基因有一定的难度。为了充分发掘Bt菌株中cry1类基因资源,本研究建立了一个高分辨率的筛选cry1类基因的PCR - RFLP方法。通过生物信息学软件对cry1类模式基因进行聚类分析,根据全长基因的5’及3’端差异将cry1类基因分成4个大类,分别设计通用引物,酶切分析只需要区分一个大类中的几个基因,提高了分辨率。该方法可以快速、准确、有效地对Bt菌株中的cry1类基因进行分型,特别是可以检测出已知及新型的cry1类基因。
通过原有的cry1类基因PCR - RFLP鉴定方法和新建立的体系对3株含有多种cry1类基因的Bt菌株进行了鉴定,发现原有体系在cry1类基因较多时无法准确检测,而且不能确定是否含有cry1I类基因,而新的体系对3株Bt菌株都达到了准确鉴定的目的,并且发现了一些国内还没有报道过的基因型,如cry1Hb、cry1Ja、cry1Ka、cry1La等。新体系不仅适用于单基因样品的分型,也适用于一株菌株中含有多个基因的样品的分型。
应用新建立的平台对72株苏云金芽胞杆菌标准菌株和86株Bt野生菌株进行cry1类基因型的鉴定,发现标准菌株中有11株含有cry1类基因,而野生菌株中绝大部分都含有cry1类基因,从中选取了19个cry1新基因。另外根据Bt标准菌株T03B001的全基因组测序结果,发现了两个cry1新基因。
根据这些基因的全长序列设计扩增cry1类基因的全长引物,成功克隆上述基因,除cry1Na外,20个cry1基因被国际Bt命名委员会正式命名为cry1Aa16、cry1Ab23、cry1Ab24、cry1Ah3、cry1Ai2、cry1Ba7、cry1Bb2、cry1Be4、cry1Ca12、cry1Da3、cry1Ea9、cry1Fa3、cry1Fa4、cry1Hb2、cry1Ia19、cry1Ib5、cry1Ie2、cry1Ja2、cry1Ka2、cry1La2。利用表达载体pEB在大肠杆菌Rosetta(DE3)中对这些基因进行表达,表达产物为60 - 130 kDa左右的蛋白。除Cry1Ka2和Cry1La2两种蛋白外,其它的Cry1蛋白都对敏感小菜蛾有毒力。没有活性报道的Cry1Hb蛋白对小菜蛾和棉铃虫都有毒力,LC50分别为305.82(241.66 - 391.76)μg/mL和315.12(225.68 - 456.86)μg/mL。Cry1Ba7和Cry1Ea9两种蛋白对大猿叶甲有较高的毒力,LC50分别为5.21(3.84 - 8.65)μg/mL和55.78(43.40 - 88.42)μg/mL,其中Cry1Ea对鞘翅目害虫的活性未见报道。Cry1Ca12对甜菜夜蛾有较高的毒力,LC50为89.11(60.54 - 121.26)×10-3μg/mL。Cry1Ie2和Cry1Na两种蛋白对亚洲玉米螟有较高的毒力,LC50分别为0.12(0.09 - 0.15)μg/mL和0.15(0.12 - 0.18)μg/mL。
Bacillus thuringiensis is the most widely applied type of microbial pesticides because its high specificity and environmental safety. The primarily insecticidal protein was Insecticidal Crystal proteins (ICPs)which were encoded by cry or cyt genes. The cry1-type genes were promising tools for effectively controling many species of important lepidopteran pests, but it is difficult to identify known and unknown cry1-type genes rapidly and accurately because of the limitations of identify methods. In order to identify more cry1-type genes, we established a PCR - RFLP identification method of cry1-type genes with high resolution. According to the difference of cry1-type genes’5’and 3’ends, they were divided into four categories and four pairs of universal primers were designed respectively, so the cry1-type genes in the same category could be determined by analysis of RFLP and the resolution is higher. This method could identify cry1-type genes of Bt strains fast rapidly, accurately and effectively, especially identify both known and novel cry1-type genes.
By using the original method and new method, three Bt strains which contain many kinds of cry1-type genes were identified respectively. The results showed that the original method could not determine cry1-type genes of Bt strains which contain many genes, and could not determine whether Bt strains contain cry1I genes. The new method could identify three Bt strains which contain many kinds of cry1-type genes accurately, and found some genes that did not find in China, for example, cry1Hb, cry1Ja, cry1Ka, cry1La, and so on. The new method suited for Bt strains which contains one cry1-type gene or many kinds of cry1-type genes.
In the identification of the 72 standard strains and 86 Bt isolates, we found 11 standard strains contained cry1-type genes and almost all Bt isolates contained cry1-type genes, and 19 of them were novel. We found other two novel cry1-type genes from Bt standard strains by whole genome sequencing.
According to known cry1-type genes’sequences, we designed full-length primers and successfully cloned them except cry1Na, they were designated as cry1Aa16, cry1Ab23, cry1Ab24, cry1Ah3, cry1Ai2, cry1Ba7, cry1Bb2, cry1Be4, cry1Ca12, cry1Da3, cry1Ea9, cry1Fa3, cry1Fa4, cry1Hb2, cry1Ia19, cry1Ib5, cry1Ie2, cry1Ja2, cry1Ka2, cry1La2 by International Nomenclature Committee of Bt. They were successfully expressed in E.coli (Rosetta) by pEB expression vector, expressed products were 60 - 130 kDa. Cry1 protains had insecticidal activity against Plutella xylostella except Cry1Ka2 and Cry1La2. The insecticidal activity of Cry1Hb had not report, but we found it had activity against Plutella xylostella and Heliothis armigera, and the LC50 were 305.82 (241.66 - 391.76)μg/ml and 315.12 (225.68 - 456.86)μg/ml. Cry1Ba7 and Cry1Ea9 had insecticidal activity against Colaphellus bowringi, the LC50 were 5.21 (3.84 - 8.65)μg/ml and 55.78 (43.40 - 88.42)μg/ml, and the activity of Cry1Ea9 against Copeoptera pests was reported firstly. Cry1Ca12 had insecticidal activity against Spodoptera exigua, the LC50 were 89.11 (60.54 - 121.26)×10-3μg/ml. Cry1Ie2 and Cry1Na had insecticidal activity against Ostrinia furnacalis, the LC50 were 0.12 (0.09 - 0.15)μg/ml and 0.15 (0.12 - 0.18)μg/ml.
通过原有的cry1类基因PCR - RFLP鉴定方法和新建立的体系对3株含有多种cry1类基因的Bt菌株进行了鉴定,发现原有体系在cry1类基因较多时无法准确检测,而且不能确定是否含有cry1I类基因,而新的体系对3株Bt菌株都达到了准确鉴定的目的,并且发现了一些国内还没有报道过的基因型,如cry1Hb、cry1Ja、cry1Ka、cry1La等。新体系不仅适用于单基因样品的分型,也适用于一株菌株中含有多个基因的样品的分型。
应用新建立的平台对72株苏云金芽胞杆菌标准菌株和86株Bt野生菌株进行cry1类基因型的鉴定,发现标准菌株中有11株含有cry1类基因,而野生菌株中绝大部分都含有cry1类基因,从中选取了19个cry1新基因。另外根据Bt标准菌株T03B001的全基因组测序结果,发现了两个cry1新基因。
根据这些基因的全长序列设计扩增cry1类基因的全长引物,成功克隆上述基因,除cry1Na外,20个cry1基因被国际Bt命名委员会正式命名为cry1Aa16、cry1Ab23、cry1Ab24、cry1Ah3、cry1Ai2、cry1Ba7、cry1Bb2、cry1Be4、cry1Ca12、cry1Da3、cry1Ea9、cry1Fa3、cry1Fa4、cry1Hb2、cry1Ia19、cry1Ib5、cry1Ie2、cry1Ja2、cry1Ka2、cry1La2。利用表达载体pEB在大肠杆菌Rosetta(DE3)中对这些基因进行表达,表达产物为60 - 130 kDa左右的蛋白。除Cry1Ka2和Cry1La2两种蛋白外,其它的Cry1蛋白都对敏感小菜蛾有毒力。没有活性报道的Cry1Hb蛋白对小菜蛾和棉铃虫都有毒力,LC50分别为305.82(241.66 - 391.76)μg/mL和315.12(225.68 - 456.86)μg/mL。Cry1Ba7和Cry1Ea9两种蛋白对大猿叶甲有较高的毒力,LC50分别为5.21(3.84 - 8.65)μg/mL和55.78(43.40 - 88.42)μg/mL,其中Cry1Ea对鞘翅目害虫的活性未见报道。Cry1Ca12对甜菜夜蛾有较高的毒力,LC50为89.11(60.54 - 121.26)×10-3μg/mL。Cry1Ie2和Cry1Na两种蛋白对亚洲玉米螟有较高的毒力,LC50分别为0.12(0.09 - 0.15)μg/mL和0.15(0.12 - 0.18)μg/mL。
Bacillus thuringiensis is the most widely applied type of microbial pesticides because its high specificity and environmental safety. The primarily insecticidal protein was Insecticidal Crystal proteins (ICPs)which were encoded by cry or cyt genes. The cry1-type genes were promising tools for effectively controling many species of important lepidopteran pests, but it is difficult to identify known and unknown cry1-type genes rapidly and accurately because of the limitations of identify methods. In order to identify more cry1-type genes, we established a PCR - RFLP identification method of cry1-type genes with high resolution. According to the difference of cry1-type genes’5’and 3’ends, they were divided into four categories and four pairs of universal primers were designed respectively, so the cry1-type genes in the same category could be determined by analysis of RFLP and the resolution is higher. This method could identify cry1-type genes of Bt strains fast rapidly, accurately and effectively, especially identify both known and novel cry1-type genes.
By using the original method and new method, three Bt strains which contain many kinds of cry1-type genes were identified respectively. The results showed that the original method could not determine cry1-type genes of Bt strains which contain many genes, and could not determine whether Bt strains contain cry1I genes. The new method could identify three Bt strains which contain many kinds of cry1-type genes accurately, and found some genes that did not find in China, for example, cry1Hb, cry1Ja, cry1Ka, cry1La, and so on. The new method suited for Bt strains which contains one cry1-type gene or many kinds of cry1-type genes.
In the identification of the 72 standard strains and 86 Bt isolates, we found 11 standard strains contained cry1-type genes and almost all Bt isolates contained cry1-type genes, and 19 of them were novel. We found other two novel cry1-type genes from Bt standard strains by whole genome sequencing.
According to known cry1-type genes’sequences, we designed full-length primers and successfully cloned them except cry1Na, they were designated as cry1Aa16, cry1Ab23, cry1Ab24, cry1Ah3, cry1Ai2, cry1Ba7, cry1Bb2, cry1Be4, cry1Ca12, cry1Da3, cry1Ea9, cry1Fa3, cry1Fa4, cry1Hb2, cry1Ia19, cry1Ib5, cry1Ie2, cry1Ja2, cry1Ka2, cry1La2 by International Nomenclature Committee of Bt. They were successfully expressed in E.coli (Rosetta) by pEB expression vector, expressed products were 60 - 130 kDa. Cry1 protains had insecticidal activity against Plutella xylostella except Cry1Ka2 and Cry1La2. The insecticidal activity of Cry1Hb had not report, but we found it had activity against Plutella xylostella and Heliothis armigera, and the LC50 were 305.82 (241.66 - 391.76)μg/ml and 315.12 (225.68 - 456.86)μg/ml. Cry1Ba7 and Cry1Ea9 had insecticidal activity against Colaphellus bowringi, the LC50 were 5.21 (3.84 - 8.65)μg/ml and 55.78 (43.40 - 88.42)μg/ml, and the activity of Cry1Ea9 against Copeoptera pests was reported firstly. Cry1Ca12 had insecticidal activity against Spodoptera exigua, the LC50 were 89.11 (60.54 - 121.26)×10-3μg/ml. Cry1Ie2 and Cry1Na had insecticidal activity against Ostrinia furnacalis, the LC50 were 0.12 (0.09 - 0.15)μg/ml and 0.15 (0.12 - 0.18)μg/ml.
引文
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Rajamohan F, Lee M K, Dean D H. 1998. Bacillus thuringiensis insecticidal proteins: molecular mode of action. J Prog Nucleic Acid Res Mol Biol, 60: 1~27.
Rasko D A, Alther M R, Han C S. 2005. Genomics of the Bacillus cereus group of organisms. J FEMS Microbiol Rev, 29: 303 ~ 329.
Rodriguez P C, Galan W L, Barjac H. 1990. Bacillus thuringiensis subspecies neoleonensis serotype H-24, a new subspecies which produces a triangular crystal. J Invertebr Pathol1 Sep, 56(2): 280~284.
Schnepf E, Crickmore N, VanRie J, et al. 1998. Bacillus thuringiensis and its pesticidal crystal Proteins. J Microbiol Mol Biol Rev, 62: 775~806.
Schnepf H E, Whiteley H R. 1981. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. J Proceedings of the National Academy of Sciences of the United States of America, 78: 2893~2897.
Schuler T H, Poppy M G, Kerry, B R. 1998. Insect-resistant transgenic plants. J Trends in Biotechnology, 16: 168~175.
Shu C L, Yan G X, Wang R Y, et al. 2009. Characterization of a novel cry8 gene specific to Melolonthidaepests: Holotrichia oblita and Holotrichia parallela. J Applied and Environmental Microbiology, 97: 123~129.
Song F P, Zhang J, Gu A, et al. 2003. Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene. J Applied and Environmental Microbiology, 69: 5207~5211.
Swiecicka I, Fiedoruk K, Bednarz G. 2002. The occurrence and properties of Bacillus thuringiensis isolated from free-living animals. J Lett Appl Microbiol, 34(3): 194~198.
Tabashnik B E. 1997. One gene in diamondback moth confers resistance to four Bacillus thuringiensis toxins. J Agriculture Sciences, 94: 1640~1644.
Tabashnik B E, Carriere Y, Dennehy T J. 2003. Insect resistance to transgenic Bt crops: lessons from the laboratory and field. J Journal of Economic Entomology, 96: 1031~1038.Tabashnik B E, Cushing N L, Finson N. 1990. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J Journal of Economic Entomology, 83: 1671~1676.
Tabashnik B E, Dennehy T J, Sims M A. 2002. Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab. J Applied and Environmental Microbiology, 68: 3790~3794.
Tabashnik B E, Gassmann A J, Crowder D W. 2008. Insect resistance to Bt crops: evidence versus theory. J Nature Biotechnology, 26: 199~202.
Tabashnik B E, VanRensburg J B, Carrère Y. 2009. Field-evolved insect resistance to Bt crops: definition, theory, and data. J Econ Entomol 102: 2011~2025.
Tojo A, Aizawa K. 1983. Dissolution and degradation of Bacillus thuringiensisδ-endotoxin by gut juice protease of the silkworm Bombyx mori. J Applied and Environmental Microbiology, 45: 576~580.
Vaeck M, Reynaerts A, Hofte H. 1987. Transgenic plants protected from insect attack. J Nature, 328: 33~37.
Valaitis A P, Augustin S, Clancy K M. 1999. Purification and characterization of western spruce bud worm larval midgut proteinases and comparison of gut activation of laboratory reared and field collected insects. J Insect Biochem. Mol. Biol. 29: 405-415.
Wei J Z, HaleK L, Carta E. 2003. Bacillus thuringiensis crystal proteins that target nematodes. J Proc Natl Acad Sci USA, 100: 2760~2765.
Xu Y, Nagai M, Bagdasarian M, et al. 2001. Expression of the p20 gene from Bacillus thuringiensis H-14 increases Cry11A toxin production and enhances mosquito-larvicidal activity in recombinant gram-negative bacteria. J Applied and Environmental Microbiology, 67: 3010-3015.
Zhang L, Lin J, Luo L, et al. 2007. A novel Bacillus thuringiensis strain LLB6 isolated from bryophytes, and its new cry2Ac-type gene. J Letters in Applied Microbiology, 44(3): 301~307.
Zhao J Z, Cao J, Collins H L, et al. 2005. Concurrent use of transgenic plants expressing a single and two Bacillus thuringiensis genes speeds insect adaptation to pyramided plants. J Proc Natl Sci USA, 102(24): 8426~8430.