少孢节丛孢菌几丁质酶AO-492对线虫的降解作用研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0010

摘要/Abstract

摘要：

【目的】为探究捕食线虫真菌少孢节丛孢菌几丁质诱导过程中分泌蛋白AO-492的生物学功能。【方法】对少孢节丛孢菌几丁质酶AO-492主要结构域编码区进行基因克隆及分子特征分析，并在毕赤酵母中进行表达。利用镍柱亲和层析法纯化重组蛋白ReAO-Z492，采用NAG检测法分析了该重组蛋白在不同温度、pH及金属离子条件下的酶学活性，并将其作用于秀丽隐杆线虫及虫卵分析其生物学功能。【结果】几丁质酶AO-492有信号肽，无跨膜结构域，含有两个几丁质结合结构域和一个糖苷水解酶18家族结构域，并含有糖苷水解酶18家族几丁质酶高度保守的底物结合位点-SVGGWT-和水解酶活性位点-FDGGDLDWE-，含有典型的TIM桶形分子结构。系统进化分析显示，该蛋白与坚粘孢单顶孢几丁质酶（EPS35099.1）的亲缘关系相对最近。SDS-PAGE和Western Blot分析表明，ReAO-Z492分子量约为59 kD，可与小鼠抗少孢节丛孢菌多克隆抗体发生特异性反应。ReAO-Z492最适温度为40℃，最适pH为7.0；Mg²⁺对其酶活有促进作用，而Ag⁺、Cu²⁺、Fe³⁺和Zn²⁺有抑制作用。ReAO-Z492对秀丽隐杆线虫体壁及其虫卵卵壳有较强的降解活性。【结论】少孢节丛孢菌几丁质酶 AO-492 对秀丽隐杆线虫及虫卵具有较强的降解作用。

关键词: 少孢节丛孢菌, 几丁质酶, ReAO-Z492, 酶学活性, 捕食线虫真菌

Abstract:

【Objective】To explore the biological function of secreted protein AO-492 in the process of chitin induction from nematode-trapping fungus Arthrobotrys oligospora.【Method】The coding region of the main domain of chitinase AO-492 from A. oligospora was cloned and analyzed, and expressed in Pichia pastoris. The recombinant protein ReAO-Z492 was purified by nickel column affinity chromatography. The enzymatic activity of the recombinant protein under different temperature, pH and metal ion conditions was analyzed by NAG assay, and its biological function was analyzed by acting on Caenorhabditis elegans and eggs.【Result】The chitinase AO-492 had a signal peptide, no transmembrane domain, contained two chitin binding domains and a glycoside hydrolase 18 family domain, and the highly conserved substrate binding site of the glycoside hydrolase 18 family chitinase -SVGGWT- and the hydrolase active site -FDGGDLDWE-, as well as a typical TIM barrel molecular structure. Phylogenetic analysis showed that the protein had the closest genetic relationship with chitinase（EPS35099.1）. SDS-PAGE and Western Blot analysis showed that the molecular weight of ReAO-Z492 was about 59 kD, which specifically reacted with mouse polyclonal antibody against A. oligospora. The optimal temperature and pH of ReAO-Z492 were 40℃ and 7.0, respectively. Mg²⁺ promoted the enzyme activity, while Ag⁺, Cu²⁺, Fe³⁺ and Zn²⁺ inhibited it. ReAO-Z492 had strong degrading activity on the body wall and egg shell of C. elegans.【Conclusion】The chitinase AO-492 of A. oligospora has a strong degradation effect on C. elegans and its eggs.

Key words: Arthrobotrys oligospora, chitinase, ReAO-Z492, enzymatic activity, nematode-trapping fungi

张嘉华, 张慧梅, 马喜喜, 孙焱森, 李若冰, 李柠杏, 才学鹏, 乔军, 孟庆玲. 少孢节丛孢菌几丁质酶AO-492对线虫的降解作用研究[J]. 生物技术通报, 2024, 40(5): 261-268.

ZHANG Jia-hua, ZHANG Hui-mei, MA Xi-xi, SUN Yan-sen, LI Ruo-bing, LI Ning-xing, CAI Xue-peng, QIAO Jun, MENG Qing-ling. Degradation of Nematodes by Chitinase AO-492 from Arthrospora oligospora[J]. Biotechnology Bulletin, 2024, 40(5): 261-268.

图/表 10

表1 本研究所用引物信息

Table 1 Primers’ information used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
492-F 492-R 492-ZF 492-ZR	ATGAGATCTCTGTTTCTACGAG TCAACAGTCTCTCGGCAGTAAG GCTACGTAATGCATCATCATCATCATCATAGATCTCTGTTTCTACGA GCGGCCGCTCAACAGTCTCTCGGCAGTAAG	1 620 1 654

图1 少孢节丛孢菌Xj-2 AO-492基因的PCR扩增 M：DL2000 DNA marker；1：阴性对照；2-3：A0-492基因扩增产物

Fig. 1 PCR amplification of AO-492 gene of A. oligospora Xj-2 strain M: DL2000 DNA marker; 1: negative control; 2-3: amplified products of AO-492 gene

图2 少孢节丛孢菌几丁质酶AO-492的结构域模式图

Fig. 2 Domain pattern of chitinase AO-492 from A. oligospora

图3 几丁质酶AO-492遗传进化分析

Fig. 3 Genetic evolution analysis of chitinase AO-492

图4 重组质粒pPIC9K-AO492的双酶切鉴定 M：DL8000 DNA marker；1：未酶切的pPIC9K-AO492；2-3：pPIC9K-AO492双酶切产物

Fig. 4 Identification of recombinant plasmid pPIC9K-AO492 by double-nzyme digestion M: DL8000 DNA marker; 1: undigested pPIC9K-AO492; 2-3: double-enzyme digested products of pPIC9K-AO492

图5 重组酵母菌株的筛选 A：MD平板；B：含1 mg/mL G418的YPD培养基；C：含2 mg/mL G418的YPD培养基；D：含3 mg/mL G418的YPD培养基

Fig. 5 Screening of recombinant yeast strains A: MD plate; B: YPD medium containing 1 mg/mL G418; C: YPD medium containing 2 mg/mL G418; D: YPD medium containing 3 mg/mL G418

图6 重组蛋白ReAO-Z492的SDS-PAGE （A）及Westem blot（B）鉴定 M：蛋白分子质量标准；（A）1：pPIC9K空载体甲醇诱导72 h的培养液上清；2-6：甲醇分别诱导24、48、72、96和120 h的培养液上清；（B）1-2：纯化后浓缩的ReAO-Z492

Fig. 6 Identification of recombinant protein ReAO-Z492 by SDS-PAGE (A) and Western blot (B) M: Protein molecular quality standard ; 1: culture supernatant of pPIC9K empty vector induced by methanol for 72 h ; 2-6: culture supernatants induced by methanol for 24,48,72,96 and 120 h, respectively. (B) 1-2: Purified and concentrated ReAO-Z492

图7 重组几丁质酶AO-492相对酶活的测定 A：NAG标准曲线；B:不同pH对AO-492酶活性的影响；C：不同温度对AO-492酶活性的影响；D：不同金属离子对AO-492酶活性的影响。****表示P≤0.0001，ns表示P>0.05

Fig. 7 Determination of relative enzymatic activity of recombinant chitinase AO-492 A: NAG standard curve. B: The effect of different pH on the activity of AO-492. C: The effect of different temperatures on the activity of AO-492. D: The effect of different metal ions on the activity of AO-492. * * * * indicates P ≤ 0.0001, ns indicates P > 0.05

图8 ReAO-Z492对秀丽隐杆线虫的降解作用对照组：A1-A3：用灭活的ReAO-Z492处理线虫0、3、6 h；试验组：B1-B3、C1-C3和D1-D3：ReAO-Z492处理线虫0、3、6 h。箭头所指为线虫部分体壁降解变化部位

Fig. 8 Degradation effect of ReAO-Z492 on C. elegans Control: A1-A3: C. elegans were treated with inactivated ReAO-Z492 for 0, 3, and 6 h. Experimentl: B1-B3, C1-C3 and D1-D3: C. elegans were treated with ReAO-Z492 for 0, 3, and 6 h. The arrow refers to the part of the body wall degradation of nematodes

图9 ReAO-Z492对秀丽隐杆线虫虫卵的降解作用对照组：A1-A3：用灭活的ReAO-Z492处理虫卵0、6、12 h；试验组：B1-B3：ReAO-Z492处理虫卵0、6、12 h。箭头所指为虫卵卵壳降解部分

Fig. 9 Degradation effect of ReAO-Z492 on C. elegans eggs Control: A1-A3: C. elegans eggs were treated with inactivated ReAO-Z492 for 0, 6, and 12 h. Experiment: B1-B3: C. elegans eggs were treated with ReAO-Z492 for 0, 6, and 12 h. The arrow refers to the degradation part of egg shell

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