苏云金芽胞杆菌可视化快速表达载体的构建与特性分析

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

摘要/Abstract

摘要：

【目的】构建可以快速高效表达苏云金芽胞杆菌（Bacillus thuringiensis，Bt）杀虫基因的表达载体，提高Bt杀虫基因发掘及功能研究效率。【方法】以pUC18载体为基础，构建一个以cry1Ac基因启动子p1Ac指导、融合绿色荧光蛋白GFP的可视化快速表达载体p1Ac-GFP，并从生物活性、碱溶性、抗胰蛋白酶稳定性、培养条件等方面对其进行分析。【结果】p1Ac-GFP在大肠杆菌中指导表达的Cry1Ac蛋白与Bt来源的蛋白在杀虫活性方面无明显差异。同时，p1Ac-GFP表达的Cry1Ac蛋白能够溶解于50 mmol/L Na₂CO₃溶液中，可溶性组分可被胰蛋白酶消化为60 kD大小的核心活性片段。此外，p1Ac-GFP大肠杆菌宿主菌株可以发出绿色荧光，培养菌液荧光强度与所表达的Bt蛋白浓度呈较好的线性关系。培养条件优化结果显示，37℃、培养液与装瓶体积比为5/5、48 h的培养时间最适合p1Ac-GFP表达Bt Cry1Ac蛋白。【结论】p1Ac-GFP表达Bt蛋白时可以直接使用菌液荧光强度作为生物活性测定的浓度指示依据，有助于Bt基因杀虫活性功能的快速批量筛选。

关键词: 苏云金芽胞杆菌, 杀虫基因, cry1Ac启动子, 大肠杆菌, 表达载体

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

张静安, 胡孝龙, 曹蓓蓓, 廖敏, 束长龙, 张杰, 王奎, 操海群. 苏云金芽胞杆菌可视化快速表达载体的构建与特性分析[J]. 生物技术通报, 2025, 41(1): 95-102.

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.

图/表 7

图1 p1Ac-GFP载体的构建图 a：载体构建图；b：linker序列；c：转化TOP10菌液示意图

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

表1 p1Ac-GFP表达条件优化设计

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

图2 Cry1Ac蛋白的SDS-PAGE分析 M：Marker；1：p1Ac-GFP空载体蛋白（阴性对照）；2-5：p1Ac-GFP表达的Cry1Ac蛋白（分别为菌液，超声破碎菌液、上清、沉淀）；6：HD73菌株表达的Cry1Ac蛋白（阳性对照）

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）

表2 Cry1Ac蛋白对小菜蛾生物活性测定结果

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

图3 Cry1Ac蛋白的溶解性与酶解性 M：Marker；1：HD73表达的Cry1Ac蛋白（碱溶后可溶组分）；2：HD73表达的Cry1Ac蛋白（胰蛋白酶活化）；3：p1Ac-GFP表达的Cry1Ac蛋白（碱溶后可溶组分）；4：p1Ac-GFP表达的Cry1Ac蛋白（胰蛋白活化）；5：p1Ac-GFP表达的Cry1Ac蛋白（碱溶后不可溶组分）

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）

图4 不同蛋白浓度下荧光强度值

Fig. 4 Fluorescence intensity values at different protein concentrations

图5 p1Ac-GFP表达Cry1Ac蛋白的条件优化 a：20℃下不同培养时间与装液量对菌液荧光强度与OD600nm的影响；b：30℃下不同培养时间与装液量对菌液荧光强度与OD600nm的影响；c：37℃下不同培养时间与装液量对菌液荧光强度与OD600nm的影响

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℃

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