一种新型的曲霉属洁净可逆性诱导表达系统

李宝意; 周胜敏

doi:10.14135/j.cnki.1006-3080.20220407001

一种新型的曲霉属洁净可逆性诱导表达系统

doi: 10.14135/j.cnki.1006-3080.20220407001

李宝意,
周胜敏^,

华东理工大学生物反应器工程国家重点实验室, 上海200037

基金项目: 国家重点研发计划政府间国际科技创新合作重点专项 (2017YFE0129600)

详细信息

作者简介:
李宝意（1991—），女，安徽芜湖人，硕士生，主要研究方向：基础生物学、合成生物学、化学生物学。E-mail：476089724@qq.com

通讯作者:
周胜敏， E-mail：zhoushengmin@ecust.edu.cm

中图分类号: Q78
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出版历程
- 收稿日期: 2022-04-07
- 网络出版日期: 2022-09-06

Construction and Optimization of a Novel Expression System Using H₂O₂as Inducer in Aspergillus

Li Baoyi,
Zhou Shengmin^,

The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

摘要

摘要: 构建了一种以构巢曲霉为表达宿主、AnPrxA启动子为表达用启动子、过氧化氢为表达诱导剂的新型高效曲霉表达体系。同时在双氧水诱导和非诱导条件下，评估构巢曲霉过氧化物氧还酶编码基因AnPrxA含启动子（P_PRX）的上游调控区介导绿色荧光蛋白（GFP）表达的特征。利用合成生物学手段构建4个含不同长度启动子（P_PRX）表达盒的克隆载体并转化尿嘧啶生物合成缺陷型构巢曲霉；分析了AnPrxA基因上游2033 bp序列中潜在的转录调控元件；利用荧光定量PCR测定表达盒拷贝数；考察与P_PRX融合的GFP表达情况；并且探讨双氧水诱导浓度和作用时间。结果表明，AnPrxA基因翻译起始密码子上游2033 bp序列、且至少包含9种真菌中常见的基因表达调控元件，其大多数呈多拷贝排列；虽然上游2033 bp的区域对gfp的转录均有贡献，但就GFP蛋白表达而言，AnPrxA上游500 bp的区域已足够；双氧水浓度范围0.5~2.0 mmol/L的诱导条件下，相对对照值，P_PRX介导GFP表达的最大诱导比率为3倍，与先前鉴定的2种曲霉属双氧水诱导型启动子相似，但目标蛋白的相对表达强度高于两者。鉴于P_PRX介导gfp转录的诱导比率（30倍）显著高于其蛋白表达的诱导比率（3倍），推测目的基因5’端编码区的部分序列可能影响蛋白质的翻译。
- PRX /
- 蛋白表达 /
- 构巢曲霉 /
- 过氧化氢 /
- 表达调控
Abstract: Aspergillus represents a potential host for expression of recombinant proteins after bacteria, yeast, plant and animal cells. Due to the late start of Aspergillus expression system research, the technology is still immature. And the number of alternative expression vectors and available promoters are limited, which limits the application of Aspergillus expression system. On our previous work, we found a peroxiredoxin from Aspergillus nidulans (AnPrxA) could be expressed at a high level and its expression level responded to H₂O₂ in the medium. Based on the low price and self-decomposition properties of H₂O₂, we explored a novel and efficient A. nidulans expression system using A. nidulans as the expression host, AnPrxA promoter as the expression promoter, and H₂O₂ as the expression inducer. The specific research is as follows. Four promoters of different lengths of the 5’untranslated regions (5’-UTR) of A. nidulans (P_PRX500, P_PRX1000, P_PRX1500 and P_PRX2033) were amplified and cloned in vectors, and the ability of the four promoters to drive GFP expression was evaluated. The results depicted that all four expression systems were sensitive to H₂O₂, and P_PRX500 was sufficient for GFP expression, but the induction ratio of P_PRX2033 is higher. The optimal induction time of H₂O₂ was determined to be 8 hours. The optimal induction concentration range was 0.5-2.0 mmol/L. Comparison of the conditions with and without the inducer revealed that P_prx2033 mediated a 30-fold induction ratio of GFP at the transcriptional level and a 3-fold induction ratio at the protein expression level. In the induction phase of this expression system, the residual concentration could no longer be detected after 2 hours of adding 2 mmol/L H₂O₂ in the medium, and could no longer be detected after 1 hour of adding 0.5 mmol/L H₂O₂.
- PRX /
- Protein expression /
- Aspergillus nidulans /
- H₂O₂ /
- Expression regulation

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图 1 过氧化物氧还酶基因启动子介导的绿色荧光蛋白报告基因表达系统的构建

Figure 1. Construction of GFP reporter gene expression system mediated by the peroxiredoxin gene promoter

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图 2 pP_PRX-GFP系列重组质粒的酶切鉴定

Figure 2. Identification of recombinant plasmids of the pP_PRX-GFPs by digestion

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图 3 过氧化物氧还酶基因起始密码子上游序列中的潜在调控元件

Figure 3. Potential regulatory elements in the sequence of the PRX promoter of the A. nidulans

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图 4 绿色荧光蛋白报告基因表达盒整合拷贝与表达量的对应关系

Figure 4. Correspondence between GFP copy number and Specific fluorescence

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图 5 过氧化物氧还酶基因4种长度的启动子序列在诱导和非诱导条件下介导绿色荧光蛋白报告基因表达的特征

Figure 5. Characteristics of four lengths of promoter sequences of PRX mediating GFP expression under inducible and non-inducible conditions

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图 6 过氧化物氧还酶基因启动子序列在双氧水诱导和非诱导条件下介导绿色荧光蛋白报告基因表达的比较

Figure 6. Comparison of PRX promoter sequences mediating green fluorescent protein reporter gene expression under H₂O₂ induced and non-induced conditions

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图 7 双氧水不同浓度对绿色荧光蛋白报告基因表达的诱导特性。

Figure 7. Effect of GFP expression under different concentrations of H₂O₂

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表 1 本文所用的PCR扩增引物

Table 1. Primers used in this study

Primer	Primer base sequences(5’Š3’)
F_pyrG	ATTCCCCGCGGGAATTCGATACCTGTCG
R_pyrG	GCAATCCGCGGTCAGTGCTTGTCTACCAG
P1-prx2033	TTTTGCCCTTGCTGCCTC
P2-prx2033	CGGGGGATCCACTAGTGGTTACGACTTCTGGAAAAC
P1-prx1500	CAGGGGCTCGAAAACTG
P2-prx1500	CGGGGGATCCACTAGTATAGATAGTATTCCAGGTTGATTTCG
P1-prx1000	AACACGGATGCCAACTGC
P2-prx1000	CGGGGGATCCACTAGTCTATTAAGGATACGTCTGGC
P1-prx500	CGTCGCTCGAAGTAGCAG
P2-prx500	CGGGGGATCCACTAGTCGGCGATGTGACAAACAG
P3-prx	GTTCTTCTCCTTTACTCATTGTGTAAGATAGTGGTTG
P4-prx	GGATGAACTATACAAATAATAGTGATTTAATAGCTCC
P5-prx	ACCGCGGTGGCGGCCGCAAAGAAGGATTACCTCTAAAC
P6-prx	TTCGAGATCAGGGGAGG
F_gfp	ATGAGTAAAGGAGAAGAAC
R_gfp	TTATTTGTATAGTTCATCCATGCC
F-YG-GFP	CTGTCCACACAATCTGCCCT
R-YG-GFP	TGCCATGTGTAATCCCAGCA
F-YG-ACT	AAGCCCCCATCAATCCCAAG
R-YG-ACT	TAGAGACGTAGAAGGCGGGA

下载: 导出CSV

表 2 方差分析表

Table 2. ANOVA Table

	SS	df	MS	F	P
Groups	172.0035506	3	57.33451686	4.049535963	0.050468081
Error	113.2663444	8	14.15829304
Total	285.2698949	11	25.93362681

下载: 导出CSV

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