Construction and Characterization of Rapid Visual Expression Vector for Bacillus thuringiensis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0608

Abstract

Abstract:

【Objective】The objective of this study is to develop an expression vector that enables rapid and efficient expression of insecticidal genes in Bacillus thuringiensis（Bt）, aiming to increase the efficiency of gene discovery and functional research in Bt.【Method】The pUC18 vector was employed for the construction of a rapid visual expression vector, namely p1Ac-GFP, under the guidance of the cry1Ac gene promoter p1Ac and fused with green fluorescent protein（GFP）. Subsequently, an analysis was conducted on its biological activity, alkaline solubility, anti-trypsin stability, and culture conditions.【Result】There was no statistically significant disparity in the insecticidal activity between the Cry1Ac protein expressed through p1Ac-GFP guidance in Escherichia coli and the protein derived from Bt. Meanwhile, the Cry1Ac protein expressed by p1Ac-GFP was solubilized in a 50 mmol/L Na₂CO₃ solution, and the resulting soluble fraction was enzymatically cleaved by trypsin to yield a 60 kD biologically active core fragment. Furthermore, it was observed that the host strain of p1Ac-GFP emited green fluorescence, with fluorescence intensity of the bacterial solution exhibiting a good linear relationship with expressed Bt protein concentration. The optimization results obtained from culture conditions demonstrated that optimal culture time for Bt Cry1Ac protein expression using p1Ac-GFP was 37℃ over a period of 48 h at a culture broth to bottle volume ratio of 5/5.【Conclusion】The fluorescence intensity of the bacterial solution, when p1Ac-GFP is utilized to express Bt protein, can serve as a direct indicator of concentration for biological activity assays. This facilitates the rapid verification of the insecticidal activity function of Bt genes.

Key words: Bacillus thuringiensis, insecticidal genes, cry1Ac promotor, Escherichia coli, expression vector

ZHANG Jing-an, HU Xiao-long, CAO Bei-bei, LIAO Min, SHU Chang-long, ZHANG Jie, WANG Kui, CAO Hai-qun. Construction and Characterization of Rapid Visual Expression Vector for Bacillus thuringiensis[J]. Biotechnology Bulletin, 2025, 41(1): 95-102.

Figures/Tables 7

Fig. 1 Construction diagram of p1Ac-GFP vector a: The vector construction diagram; b: the linker sequence; c: the schematic diagram of transforming TOP10 bacterial solution

Table 1 Optimized design of p1Ac-GFP expression conditions

装液量/锥形瓶体积（V/V） Liquid volume/Volume of the conical flask	20℃培养时间 Culture time for 20℃/h	装液量/锥形瓶体积（V/V） Liquid volume/Volume of the conical flask	30℃培养时间 Culture time for 30℃/h	装液量/锥形瓶体积（V/V） Liquid volume/Volume of the conical flask	37℃培养时间Culture time for 37℃/h
1/5	12	1/5	12	1/5	12
	24		24		24
	36		36		36
	48		48		48
	60		60		60
	72		72		72
2/5	12	2/5	12	2/5	12
	24		24		24
	36		36		36
	48		48		48
	60		60		60
	72		72		72
3/5	12	3/5	12	3/5	12
	24		24		24
	36		36		36
	48		48		48
	60		60		60
	72		72		72
4/5	12	4/5	12	4/5	12
	24		24		24
	36		36		36
	48		48		48
	60		60		60
	72		72		72
5/5	12	5/5	12	5/5	12
	24		24		24
	36		36		36
	48		48		48
	60		60		60
	72		72		72

Fig. 2 SDS-PAGE analysis of Cry1Ac protein M: Marker; 1: p1Ac-GFP empty vector protein（negative control）; 2-5: Cry1Ac protein expressed by p1Ac-GFP（bacterial solution, ultrasonic crushing bacterial solution, ultrasonic crushing supernatant, ultrasonic crushing precipitation, respectively）; 6: Cry1Ac protein expressed by strain HD73（positive control）

Table 2 Bioassay of Cry1Ac proteins against P. xylostella

蛋白Protein	校正死亡率±SD Corrected mortality /%
p1Ac-GFP空载体蛋白	4.60 ± 3.98^b
p1Ac-GFP Cry1Ac蛋白	100.00 ± 0.00^a
HD73 Cry1Ac蛋白	96.55 ± 3.45 ^a

Fig. 3 Solubility and enzymatic hydrolysis of Cry1Ac protein M: Marker; 1: Cry1Ac protein expressed by HD73（soluble components after dissolution）; 2: Cry1Ac protein expressed by HD73（trypsin activation）; 3: Cry1Ac protein expressed by p1Ac-GFP（soluble components after dissolution）; 4: Cry1Ac protein expressed by p1Ac-GFP（trypsin activation）; 5: Cry1Ac protein expressed by p1Ac-GFP（insoluble components after dissolution）

Fig. 4 Fluorescence intensity values at different protein concentrations

Fig. 5 Optimization of condition when Cry1Ac expressed by p1Ac-GFP a: The effects of different culture time and bottling amount on fluorescence intensity and OD600nm of bacterial solution at 20℃; b: the effects of different culture time and bottling amount on fluorescence intensity and OD600nm of bacterial solution at 30℃; c: the effects of different culture time and bottling amount on fluorescence intensity and OD600nm of bacterial solution at 37℃

References 35

[1]	彭琦, 周子珊, 张杰. 苏云金芽胞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. doi: 10.16409/j.cnki.2095-039x.2015.05.011
	Peng Q, Zhou ZS, Zhang J. Research prospects in insecticidal crystal proteins of Bacillus thuringiensis[J]. Chin J Biol Contr, 2015, 31(5): 712-722.
[2]	Caballero J, Jiménez-Moreno N, Orera I, et al. Unraveling the composition of insecticidal crystal proteins in Bacillus thuringiensis: a proteomics approach[J]. Appl Environ Microbiol, 2020, 86(12): e00476-20.
[3]	Bravo A, Gill SS, Soberón M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[J]. Toxicon, 2007, 49(4): 423-435.
[4]	Crickmore N, Berry C, Panneerselvam S, et al. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins[J]. J Invertebr Pathol, 2021, 186: 107438.
[5]	Ramalakshmi A, Udayasuriyan V, Balasubramani V. Molecular cloning of a new cry2A-type gene from Bacillus thuringiensis strain Nn10 and its expression studies[J]. Microb Pathog, 2022, 164: 105415.
[6]	Liang Z, Ali Q, Wang YJ, et al. Toxicity of Bacillus thuringiensis strains derived from the novel crystal protein Cry31Aa with high nematicidal activity against rice parasitic nematode Aphelenchoides besseyi[J]. Int J Mol Sci, 2022, 23(15): 8189.
[7]	Torres-Quintero MC, Arenas-Sosa I, Zuñiga-Navarrete F, et al. Characterization of insecticidal Cry1Cb2 protein from Bacillus thuringiensis toxic to Myzus persicae(Sulzer)[J]. J Invertebr Pathol, 2022, 189: 107731.
[8]	Peng Q, Yu QY, Song FP. Expression of cry genes in Bacillus thuringiensis biotechnology[J]. Appl Microbiol Biotechnol, 2019, 103(4): 1617-1626. doi: 10.1007/s00253-018-9552-x pmid: 30617537
[9]	郑文, 叶伟星, 彭东海, 等. 基于cry1Ac表达调控元件的苏云金芽孢杆菌表达载体构建[J]. 湖北农业科学, 2012, 51(2): 400-405.
	Zheng W, Ye WX, Peng DH, et al. Construction of Bacillus thuringiensis expression vector by using regulatory elements from cry1Ac gene[J]. Hubei Agric Sci, 2012, 51(2): 400-405.
[10]	Wang GJ, Zhang J, Song FP, et al. Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests[J]. Appl Microbiol Biotechnol, 2006, 72(5): 924-930.
[11]	Kant S, Kapoor R, Banerjee N. Identification of a catabolite-responsive element necessary for regulation of the cry4A gene of Bacillus thuringiensis subsp. israelensis[J]. J Bacteriol, 2009, 191(14): 4687-4692.
[12]	李朝睿, 杜立新, 彭琦, 等. 苏云金芽胞杆菌高效表达载体的构建[J]. 微生物学通报, 40(2): 350-361.
	Li CR, Du LX, Peng Q, et al. Construction of high-level expression vector for Bacillus thuringiensis[J]. Microbiology China, 40(2): 350-361.
[13]	杜立新. 苏云金芽胞杆菌cry8E基因转录调控机制研究[D]. 保定: 河北农业大学, 2011.
	Du LX. Study on transcriptional regulation of cry8E gene of Bacillus thuringiensis[D]. Baoding: Hebei Agricultural University, 2011.
[14]	Schnepf HE, Wong HC, Whiteley HR. Expression of a cloned Bacillus thuringiensis crystal protein gene in Escherichia coli[J]. J Bacteriol, 1987, 169(9): 4110-4118. doi: 10.1128/jb.169.9.4110-4118.1987 pmid: 3040677
[15]	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, 28(11): 192-196.
	Zhang CG, Zhao C, Shu CL, et al. Analysis of expressed Cry1Ie protein initiated by different promoters[J]. Biotechnol Bull, 2012, 28(11): 192-196.
[16]	马君兰, 束长龙, 刘东明, 等. 大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析[J]. 生物技术通报, 2011, 27(2): 80-84.
	Ma JL, Shu CL, Liu DM, et al. The nature analysis of expressed Cry1Ac protein initiated by strong promoter p1Ac from Bacillus thuringensis in E. coli[J]. Biotechnol Bull, 2011, 27(2): 80-84.
[17]	Zhou ZS, Yang SJ, Shu CL, et al. Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain[J]. J Integr Agric, 2015, 14(8): 1598-1603.
[18]	Rueden CT, Schindelin J, Hiner MC, et al. ImageJ2: ImageJ for the next generation of scientific image data[J]. BMC Bioinformatics, 2017, 18(1): 529. doi: 10.1186/s12859-017-1934-z pmid: 29187165
[19]	王建, 杨小雪, 王丹丹, 等. 对草地贪夜蛾高毒力的苏云金芽胞杆菌菌株筛选与杀虫活性研究[J]. 中国生物防治学报, 2021, 37(4): 660-670. doi: 10.16409/j.cnki.2095-039x.2021.03.008
	Wang J, Yang XX, Wang DD, et al. Screening and insecticidal activity of Bacillus thuringiensis strains with high toxicity against Spodoptera frugiperda[J]. Chin J Biol Contr, 2021, 37(4): 660-670.
[20]	Agaisse H, Lereclus D. How does Bacillus thuringiensis produce so much insecticidal crystal protein?[J]. J Bacteriol, 1995, 177(21): 6027-6032. doi: 10.1128/jb.177.21.6027-6032.1995 pmid: 7592363
[21]	Palma L, Muñoz D, Berry C, et al. Bacillus thuringiensis toxins: an overview of their biocidal activity[J]. Toxins, 2014, 6(12): 3296-3325.
[22]	Wang K, Shu CL, Zhang J. Effective bacterial insecticidal proteins against coleopteran pests: a review[J]. Arch Insect Biochem Physiol, 2019, 102(3): e21558.
[23]	Baranek J, Banaszak M, Kaznowski A, et al. A novel Bacillus thuringiensis Cry9Ea-like protein with high insecticidal activity towards Cydia pomonella larvae[J]. Pest Manag Sci, 2021, 77(3): 1401-1408. doi: 10.1002/ps.6157 pmid: 33099864
[24]	Cao BB, Shu CL, Geng LL, et al. Cry78Ba1, one novel crystal protein from Bacillus thuringiensis with high insecticidal activity against rice planthopper[J]. J Agric Food Chem, 2020, 68(8): 2539-2546.
[25]	Geng C, Liu YY, Li MM, et al. Dissimilar crystal proteins Cry5Ca1 and Cry5Da1 synergistically act against Meloidogyne incognita and delay Cry5Ba-based nematode resistance[J]. Appl Environ Microbiol, 2017, 83(18): e03505-16.
[26]	Zhang Q, Hua G, Adang MJ. Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae[J]. Insect Sci, 2017, 24(5): 714-729. doi: 10.1111/1744-7917.12401 pmid: 27628909
[27]	Panwar BS, Kaur S. Structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis[J]. J Mol Model, 2019, 25(5): 136.
[28]	Bukhari DAA, Shakoori AR. Cloning and expression of Bacillus thuringiensis cry11 crystal protein gene in Escherichia coli[J]. Mol Biol Rep, 2009, 36(7): 1661-1670. doi: 10.1007/s11033-008-9366-5 pmid: 18821029
[29]	Amadio AF, Navas LE, Sauka DH, et al. Identification, cloning and expression of an insecticide cry8 gene from Bacillus thuringiensis INTA Fr7-4[J]. J Mol Microbiol Biotechnol, 2013, 23(6): 401-409.
[30]	Sazhenskiy V, Zaritsky A, Itsko M. Expression in Escherichia coli of the native cyt1Aa from Bacillus thuringiensis subsp. israelensis[J]. Appl Environ Microbiol, 2010, 76(10): 3409-3411.
[31]	Reyaz AL, Arulselvi PI. Cloning, characterization and expression of a novel haplotype cry2A-type gene from Bacillus thuringiensis strain SWK1 native to Himalayan valley Kashmir[J]. J Invertebr Pathol, 2016, 136: 1-6. doi: 10.1016/j.jip.2016.02.005 pmid: 26906447
[32]	余宗兰, 贺利业, 龚莉, 等. 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 ZL, He LY, Gong L, et al. Cloning, expression, and biological activity of a new cry1Ie gene from Bacillus thuringiensis strain[J]. J Plant Prot, 2016, 43(2): 201-206.
[33]	张月, 李海涛, 刘荣梅, 等. 苏云金芽胞杆菌cry2Ab34基因的克隆、表达和杀虫活性分析[J]. 生物技术通报, 2017, 33(4): 185-190.
	Zhang Y, Li HT, Liu RM, et al. Cloning, expression, and insecticidal activities of gene cry2Ab34 from Bacillus thuringiensis[J]. Biotechnol Bull, 2017, 33(4): 185-190.
[34]	Pardo-López L, Soberón M, Bravo A. Bacillus thuringiensis insecticidal three-domain cry toxins: mode of action, insect resistance and consequences for crop protection[J]. FEMS Microbiol Rev, 2013, 37(1): 3-22. doi: 10.1111/j.1574-6976.2012.00341.x pmid: 22540421
[35]	Paulmurugan R, Gambhir SS. Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals[J]. Cancer Res, 2005, 65(16): 7413-7420. pmid: 16103094