长枝木霉菌几丁质酶基因克隆和抑菌活性物质结构鉴定
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摘要
长枝木霉(Trichoderma longibrachiatum)是生防木霉属真菌中的一种。长枝木霉用于植物病害的生物防治已有报道,但在东北地区对其研究还较少,它的具体的生防机制还未被揭示。几丁质酶是木霉生物防治机制中一种非常重要的胞壁降解酶,在木霉重寄生病原菌菌丝过程中起着重要的作用。同时,木霉菌产生的活性次生代谢产物,是木霉生防机制中另一重要的方面,活性次生代谢产物能够直接抑制植物病害病原菌的生长,并将其瓦解,而且与胞壁降解酶具有一定的协同拮抗作用。因此,对木霉生防机制这两方面的研究,对于进一步揭示长枝木霉的生防机制,具有非常重大的意义。
本研究首先采用分子系统学方法对1株自我采集的具有潜在生物防治东北地区多种林木、果树皮部病害和农作物病害的木霉菌进行了鉴定；然后采用特异性引物PCR、RT-PCR、RACE方法从几丁质酶诱导培养基培养的长枝木霉菌丝中克隆了几丁质酶基因；采用NCBI、SWISS-MODEL等网络工具及BioEdit、ClustalX2.0、Chimera、Cn3D等软件分析了几丁质酶的基本信息、功能位点、二级结构特征、三维结构特征等生物学特性；同时对长枝木霉菌产生的活性次生代谢产物进行了提取分离纯化与结构鉴定等研究。主要研究结果如下：
1.针对木霉菌属内不同种之间rDNA的ITS序列的相对特异性的特点,采用通用引物对木霉rDNA的ITS1+5.8S+ITS2区域以及tefl序列进行了扩增、测序,并与NCBI数据库中的木霉菌种类进行序列比对,同时应用木霉在线鉴定工具TrichoKey2.0完成了木霉菌株的鉴定,最终将自我采集分离的木霉菌株鉴定为长枝木霉菌(Trichoderma longibrachiatum T05),这一结果与以前的形态学鉴定结果完全相同。
2.采用透明圈法和DNS比色法对长枝木霉产生的几丁质酶和p-1,3-葡聚糖酶活性进行了研究,进一步证明了几丁质酶和β-1,3-葡聚糖酶为诱导酶的结论,得出了长枝木霉在诱导液体培养基中的几丁质酶和β-1,3-葡聚糖酶酶活变化曲线,为进一步克隆长枝木霉几丁质酶和β-1,3-葡聚糖酶基因全长奠定了基础,提供了其酶活达到最大表达量所需的最佳时间,为提取含有高丰度mRNA的长枝木霉总RNA做好了准备。
3.应用特异性引物PCR获得了700bp左右的几丁质酶基因序列片段,并利用获得的序列片段设计了特异性引物用于cDNA片段的扩增,再利用RT-PCR技术扩增到了长枝木霉菌几丁质酶的cDNA基因片段,然后通过RACE技术扩增获得了几丁质酶基因的3’末端及5’末端,通过序列的拼接获得了1080bp的全长cDNA基因序列,命名为TL-ch42。
4.TL-ch42基因含有1080bp的完整开放阅读框(open reading frame, ORF),起始密码子为ATG,终止密码子为TGA,5’非翻译区(5'UTR)有519bp,3’非翻译区(3'UTR)有221bp共编码359aa (amino acid);碱基组成为A22.22%、C31.76%.、24.72%、T21.30%, G+C含量占56.48%、A+T含量占43.52%。翻译的氨基酸序列组成分析结果为：Ala12.78%、Cys0.28%、Asp7.22%、Glu2.50%、Phe4.17%、Gly7.78%、His1.39%、He4.44%、Lys5.00%、Leu7.50%、Met2.22%、Asn6.67%、Pro3.89%、Gln3.06%、Arg2.50%、Ser8.61%、Thr5.56%、Val6.11%、Trp2.22%、Tyr5.83%,其中丙氨酸(Ala)含量最高,半胱氨酸(Cys)含量最低。利用ExPASy的Proteomics对TL-ch42蛋白基本信息的预测结果为：分子量为38.99kD,pI为5.12,带负电荷残基(Asp+Glu)有35个,带正电荷残基(Arg+Lys)有27个,分子式为C1752H2645N457O537S9,,总原子数为5400个,不稳定指数为28.01,属稳定蛋白,脂肪族指数为77.30,总平均疏水性为-0.192。
5.预测的TL-ch42蛋白信号肽最可能的剪切位点在22-23aa之间,为TSA-SP;该蛋白在8～27aa、36～56aa、123～141aa和201～223aa间有4个疏水区域,跨膜区预测位置也在8～27aa、36-56aa、123-141aa和201～223aa间,与亲/疏水性预测结果一致；TL-ch42蛋白的二级结构中无规则卷曲、α螺旋和β折叠的比例分别为61.84%、23.96%、14.21%。对该蛋白进行亚细胞定位,发现TL-ch42的蛋白主要位于溶酶体、胞外、过氧化物酶体、内质网隔膜及内质网管腔；用SWISS-MODEL在线三维结构比对预测,用Chimera和Cn3D输出了几丁质酶的三维模型。
6.运用薄层层析、柱层析、高效液相色谱等分离分析方法对长枝木霉发酵液中的抑菌活性物质进行了分离、纯化,采用生物活性追踪的方法,逐步明确了发酵液中起抑菌作用的成分,并对其化学结构进行了鉴定。试验结果表明,乙酸乙酯为萃取抑菌活性物质的最佳溶剂,乙酸乙酯与石油醚比例达到2：1为最佳薄层层析展开剂。薄层层析展开后利用碘和紫外灯显色,发现共有9个点。以不同配比的石油醚—乙酸乙酯作为洗脱液,经硅胶柱层析分离后得到9个组分,各组分经减压浓缩、挥干溶剂后,分别计算出了各组分的得率,并对各组分的抑菌活性进行了测定。组分Ⅵ的得率较高,为24.76%,且对供试病原菌菌落生长抑制率最高,表明抑菌活性成分主要存在于组分Ⅵ中。将组分Ⅵ上sephadex LH20柱进行纯化,以甲醇、水梯度洗脱,最终得到组分Ⅵ-1纯样品232mg。Ⅵ-1经HPLC检测分析,发现其纯度达到95%以上。通过高分辨质谱分析,推断该化合物的分子式为C28H32O8。经NIST库检查发现此化合物与化合物bislongiquinolide(长枝联喹啉内酯)的质谱图相吻合,丰度达95%。
Trichoderma longibrachiatum is one species of Trichoderma genus that has biocontrol potential. There are some reports on application of Trichoderma longibrachiatum to control plant diseases, but less study in the northeast of china, and the biocontrol mechanism is still not revealed. Chitinase is one important cell wall degrading enzyme in biocontrol mechanism of Trichoderma, plays the important role in the process of hyperparasitism on mycelium of plant pathogen. Meanwhile, the active secondary metabolites produced by Trichoderma is another important part in the biocontrol mechanism, they can inhibit the growth of plant pathogen directly, and make it collapsed, also have the synergetic antagonism with cell wall degrading enzymes. Thus, the study on the two part of Trichoderma biocontrol mechanism has great significance to reveal the biocontrol mechanism of Trichoderma longibrachiatum.
In this study, molecular phylogenetics was adopted to identify one strain of Trichoderma which has potential ability on biocontrol many plant pathogens in northeast of china. The chitinase cDNA gene was cloned from the Trichoderma longibrachiatum mycelia which cultured with chitinase induction medium by the methods of specific primer PCR, RT-PCR and RACE. The basic information of chitinase, function sites, secondary structure characteristics and3D characteristics were analyzed by the online tools such as NCBI and SWISS-MODEL, the molecular biological software such as BioEdit, ClustalX2.0, Chimera and Cn3D. Meanwhile, the active secondary metabolites produced by Trichoderma longibrachiatum T05were extracted, separated and its chemical structure was identified. The main results as follows:
1. According to the relative specific rDNA ITS sequences of Trichoderma genus, rDNA ITS1+5.8S+ITS2sequences region and tefl sequence were amplified and sequenced, and aligned with the ITS and tefl sequences of Trichoderma in GenBank of NCBI. Meanwhile, a online Trichoderma identification tool TrichoKey2.0was used for the further identification, the test strain was identified as Trichoderma longibrachiatum. The result was completely same as the morphology identification.
2. Using transparent circle method and DNS colorimetric method, the chitinase and P-1,3-glucanase activity of Trichoderma longibrachiatum T05were studied, further proved the conclusion of chitinase and P-1,3-glucanase were inducible enzyme, laid the foundation for cloning chitinase and P-1,3-glucanase complete cDNA gene, provided the optimum time that enzyme reaching the maximum expression, got the ready for extracting total RNA contained abundant mRNA.
3. Firstly, the fragment sequences of chitinase gene was obtained by specific primers PCR, then the specific primer was designed for amplifying the fragment of cDNA gene, the fragment of cDNA gene of chitinase from T. longibrachiatum T05, the3'-end and5'-end of chitinase cDNA gene were amplified by RACE, the complete cDNA gene (1080bp) was obtained by joining together with software ContigExpress, named as TL-ch42.
4. TL-ch42gene contained a full open reading frame (ORF) of1080bp in length. The start codon was ATG, the stop codon was TGA, endoded a putative protein of359amino acids. The length of5'and3'untranslatin region was519bp and221bp, respectively. The base composition was A22.22%, C31.76%, G24.72%, T21.30%, the G+C content of TL-ch42was56.48%, and the A+T content was43.52%. The analysis result of putative amino acid composition was as follows:Ala12.78%, Cys0.28%, Asp7.22%, Glu2.50%, Phe4.17%, Gly7.78%, His1.39%, Ile4.44%, Lys5.00%, Leu7.50%, Met2.22%, Asn6.67%, Pro3.89%, Gln3.06%, Arg2.50%, Ser8.61%, Thr5.56%, Val6.11%, Trp2.22%, Tyr5.83%, thereinto, the Ala content was highest, and Cys was lowest. The predicted molecular wight of putative protein was38.99kDa, and the pI was5.12. There were35amino acid residues with negative charge (Asp+Glu) and27amino acid residues with positive charge (Arg+Lys). The putative protein contained5400atoms with a molecular formula of C1752H2645N457O537S9. The protein was a stable protein with a instability index of28.01. The aliphatic index was77.30and the total average hydrophobicity is-0.192.
5. The most probable splice site of signal peptide of TL-ch42was predicted to be located between the22nd and23rd amino acid, which was recognized as TSA-SP. The protein contained4hydrophobic regions which were located between the8th-27th amino acid,36th-56th amino acid,123rd-141st amino acid,201st-223rd amino acid. The transmembrane region was also predicted to be located in these regions. The secondary structure was composed of random coil, a helix and β strands with a percentage of61.84%,23.96%and14.21%, respectively. The result of subcellular location of TL-ch42showed that this protein was mainly located in lysosome (lumen) and in outside, microbody (peroxisome) and endoplasmic reticulum (membrane). The tertiary structure of TL-ch42putative protein was predicted using SWISS-MODEL online and the structure was exported by Chimera and Cn3D.
6. Using the separation and analysis methods of TLC, column chromotography and HPLC, the antifungal secondary metabolites from the fermentation liquid of Trichoderma longibrachiatum T05. The antifungal part of secondary metabolites was determined and its chemical structure was identified with the method of biological activity tracing. The results showed that ethyl acetate was the optimum solvent to extract the antifungal secondary metabolites, petroleum ether:ethyl acetate=2:1was the optimum developer for TLC. Nine point of samples were observed under UV light and with I2. Nine components were obtained by silica column chromotography, different ratio of petroleum ether and ethyl acetate as eluent, yield rate of different component was calculated after condensing and drying solvent, and the antifungal activity was tested. The yield rate of component VI was higher, that is24.76%, and its inhibition rate on the tested plant pathogen was highest, showed that the main part of antifungal substance was in it. The232mg pure sample of component Ⅵ-1was obtained by sephadex LH20column chromotography with MeOH and water as eluent. The purity of component Ⅵ-1was above95%after HPLC analysis. After High resolution mass spectrometry determination, the deduced formula for this coumpound was C28H32O8. The mass spectrum was same as bislongiquinolide in NIST, the abundant was95%.
本研究首先采用分子系统学方法对1株自我采集的具有潜在生物防治东北地区多种林木、果树皮部病害和农作物病害的木霉菌进行了鉴定；然后采用特异性引物PCR、RT-PCR、RACE方法从几丁质酶诱导培养基培养的长枝木霉菌丝中克隆了几丁质酶基因；采用NCBI、SWISS-MODEL等网络工具及BioEdit、ClustalX2.0、Chimera、Cn3D等软件分析了几丁质酶的基本信息、功能位点、二级结构特征、三维结构特征等生物学特性；同时对长枝木霉菌产生的活性次生代谢产物进行了提取分离纯化与结构鉴定等研究。主要研究结果如下：
1.针对木霉菌属内不同种之间rDNA的ITS序列的相对特异性的特点,采用通用引物对木霉rDNA的ITS1+5.8S+ITS2区域以及tefl序列进行了扩增、测序,并与NCBI数据库中的木霉菌种类进行序列比对,同时应用木霉在线鉴定工具TrichoKey2.0完成了木霉菌株的鉴定,最终将自我采集分离的木霉菌株鉴定为长枝木霉菌(Trichoderma longibrachiatum T05),这一结果与以前的形态学鉴定结果完全相同。
2.采用透明圈法和DNS比色法对长枝木霉产生的几丁质酶和p-1,3-葡聚糖酶活性进行了研究,进一步证明了几丁质酶和β-1,3-葡聚糖酶为诱导酶的结论,得出了长枝木霉在诱导液体培养基中的几丁质酶和β-1,3-葡聚糖酶酶活变化曲线,为进一步克隆长枝木霉几丁质酶和β-1,3-葡聚糖酶基因全长奠定了基础,提供了其酶活达到最大表达量所需的最佳时间,为提取含有高丰度mRNA的长枝木霉总RNA做好了准备。
3.应用特异性引物PCR获得了700bp左右的几丁质酶基因序列片段,并利用获得的序列片段设计了特异性引物用于cDNA片段的扩增,再利用RT-PCR技术扩增到了长枝木霉菌几丁质酶的cDNA基因片段,然后通过RACE技术扩增获得了几丁质酶基因的3’末端及5’末端,通过序列的拼接获得了1080bp的全长cDNA基因序列,命名为TL-ch42。
4.TL-ch42基因含有1080bp的完整开放阅读框(open reading frame, ORF),起始密码子为ATG,终止密码子为TGA,5’非翻译区(5'UTR)有519bp,3’非翻译区(3'UTR)有221bp共编码359aa (amino acid);碱基组成为A22.22%、C31.76%.、24.72%、T21.30%, G+C含量占56.48%、A+T含量占43.52%。翻译的氨基酸序列组成分析结果为：Ala12.78%、Cys0.28%、Asp7.22%、Glu2.50%、Phe4.17%、Gly7.78%、His1.39%、He4.44%、Lys5.00%、Leu7.50%、Met2.22%、Asn6.67%、Pro3.89%、Gln3.06%、Arg2.50%、Ser8.61%、Thr5.56%、Val6.11%、Trp2.22%、Tyr5.83%,其中丙氨酸(Ala)含量最高,半胱氨酸(Cys)含量最低。利用ExPASy的Proteomics对TL-ch42蛋白基本信息的预测结果为：分子量为38.99kD,pI为5.12,带负电荷残基(Asp+Glu)有35个,带正电荷残基(Arg+Lys)有27个,分子式为C1752H2645N457O537S9,,总原子数为5400个,不稳定指数为28.01,属稳定蛋白,脂肪族指数为77.30,总平均疏水性为-0.192。
5.预测的TL-ch42蛋白信号肽最可能的剪切位点在22-23aa之间,为TSA-SP;该蛋白在8～27aa、36～56aa、123～141aa和201～223aa间有4个疏水区域,跨膜区预测位置也在8～27aa、36-56aa、123-141aa和201～223aa间,与亲/疏水性预测结果一致；TL-ch42蛋白的二级结构中无规则卷曲、α螺旋和β折叠的比例分别为61.84%、23.96%、14.21%。对该蛋白进行亚细胞定位,发现TL-ch42的蛋白主要位于溶酶体、胞外、过氧化物酶体、内质网隔膜及内质网管腔；用SWISS-MODEL在线三维结构比对预测,用Chimera和Cn3D输出了几丁质酶的三维模型。
6.运用薄层层析、柱层析、高效液相色谱等分离分析方法对长枝木霉发酵液中的抑菌活性物质进行了分离、纯化,采用生物活性追踪的方法,逐步明确了发酵液中起抑菌作用的成分,并对其化学结构进行了鉴定。试验结果表明,乙酸乙酯为萃取抑菌活性物质的最佳溶剂,乙酸乙酯与石油醚比例达到2：1为最佳薄层层析展开剂。薄层层析展开后利用碘和紫外灯显色,发现共有9个点。以不同配比的石油醚—乙酸乙酯作为洗脱液,经硅胶柱层析分离后得到9个组分,各组分经减压浓缩、挥干溶剂后,分别计算出了各组分的得率,并对各组分的抑菌活性进行了测定。组分Ⅵ的得率较高,为24.76%,且对供试病原菌菌落生长抑制率最高,表明抑菌活性成分主要存在于组分Ⅵ中。将组分Ⅵ上sephadex LH20柱进行纯化,以甲醇、水梯度洗脱,最终得到组分Ⅵ-1纯样品232mg。Ⅵ-1经HPLC检测分析,发现其纯度达到95%以上。通过高分辨质谱分析,推断该化合物的分子式为C28H32O8。经NIST库检查发现此化合物与化合物bislongiquinolide(长枝联喹啉内酯)的质谱图相吻合,丰度达95%。
Trichoderma longibrachiatum is one species of Trichoderma genus that has biocontrol potential. There are some reports on application of Trichoderma longibrachiatum to control plant diseases, but less study in the northeast of china, and the biocontrol mechanism is still not revealed. Chitinase is one important cell wall degrading enzyme in biocontrol mechanism of Trichoderma, plays the important role in the process of hyperparasitism on mycelium of plant pathogen. Meanwhile, the active secondary metabolites produced by Trichoderma is another important part in the biocontrol mechanism, they can inhibit the growth of plant pathogen directly, and make it collapsed, also have the synergetic antagonism with cell wall degrading enzymes. Thus, the study on the two part of Trichoderma biocontrol mechanism has great significance to reveal the biocontrol mechanism of Trichoderma longibrachiatum.
In this study, molecular phylogenetics was adopted to identify one strain of Trichoderma which has potential ability on biocontrol many plant pathogens in northeast of china. The chitinase cDNA gene was cloned from the Trichoderma longibrachiatum mycelia which cultured with chitinase induction medium by the methods of specific primer PCR, RT-PCR and RACE. The basic information of chitinase, function sites, secondary structure characteristics and3D characteristics were analyzed by the online tools such as NCBI and SWISS-MODEL, the molecular biological software such as BioEdit, ClustalX2.0, Chimera and Cn3D. Meanwhile, the active secondary metabolites produced by Trichoderma longibrachiatum T05were extracted, separated and its chemical structure was identified. The main results as follows:
1. According to the relative specific rDNA ITS sequences of Trichoderma genus, rDNA ITS1+5.8S+ITS2sequences region and tefl sequence were amplified and sequenced, and aligned with the ITS and tefl sequences of Trichoderma in GenBank of NCBI. Meanwhile, a online Trichoderma identification tool TrichoKey2.0was used for the further identification, the test strain was identified as Trichoderma longibrachiatum. The result was completely same as the morphology identification.
2. Using transparent circle method and DNS colorimetric method, the chitinase and P-1,3-glucanase activity of Trichoderma longibrachiatum T05were studied, further proved the conclusion of chitinase and P-1,3-glucanase were inducible enzyme, laid the foundation for cloning chitinase and P-1,3-glucanase complete cDNA gene, provided the optimum time that enzyme reaching the maximum expression, got the ready for extracting total RNA contained abundant mRNA.
3. Firstly, the fragment sequences of chitinase gene was obtained by specific primers PCR, then the specific primer was designed for amplifying the fragment of cDNA gene, the fragment of cDNA gene of chitinase from T. longibrachiatum T05, the3'-end and5'-end of chitinase cDNA gene were amplified by RACE, the complete cDNA gene (1080bp) was obtained by joining together with software ContigExpress, named as TL-ch42.
4. TL-ch42gene contained a full open reading frame (ORF) of1080bp in length. The start codon was ATG, the stop codon was TGA, endoded a putative protein of359amino acids. The length of5'and3'untranslatin region was519bp and221bp, respectively. The base composition was A22.22%, C31.76%, G24.72%, T21.30%, the G+C content of TL-ch42was56.48%, and the A+T content was43.52%. The analysis result of putative amino acid composition was as follows:Ala12.78%, Cys0.28%, Asp7.22%, Glu2.50%, Phe4.17%, Gly7.78%, His1.39%, Ile4.44%, Lys5.00%, Leu7.50%, Met2.22%, Asn6.67%, Pro3.89%, Gln3.06%, Arg2.50%, Ser8.61%, Thr5.56%, Val6.11%, Trp2.22%, Tyr5.83%, thereinto, the Ala content was highest, and Cys was lowest. The predicted molecular wight of putative protein was38.99kDa, and the pI was5.12. There were35amino acid residues with negative charge (Asp+Glu) and27amino acid residues with positive charge (Arg+Lys). The putative protein contained5400atoms with a molecular formula of C1752H2645N457O537S9. The protein was a stable protein with a instability index of28.01. The aliphatic index was77.30and the total average hydrophobicity is-0.192.
5. The most probable splice site of signal peptide of TL-ch42was predicted to be located between the22nd and23rd amino acid, which was recognized as TSA-SP. The protein contained4hydrophobic regions which were located between the8th-27th amino acid,36th-56th amino acid,123rd-141st amino acid,201st-223rd amino acid. The transmembrane region was also predicted to be located in these regions. The secondary structure was composed of random coil, a helix and β strands with a percentage of61.84%,23.96%and14.21%, respectively. The result of subcellular location of TL-ch42showed that this protein was mainly located in lysosome (lumen) and in outside, microbody (peroxisome) and endoplasmic reticulum (membrane). The tertiary structure of TL-ch42putative protein was predicted using SWISS-MODEL online and the structure was exported by Chimera and Cn3D.
6. Using the separation and analysis methods of TLC, column chromotography and HPLC, the antifungal secondary metabolites from the fermentation liquid of Trichoderma longibrachiatum T05. The antifungal part of secondary metabolites was determined and its chemical structure was identified with the method of biological activity tracing. The results showed that ethyl acetate was the optimum solvent to extract the antifungal secondary metabolites, petroleum ether:ethyl acetate=2:1was the optimum developer for TLC. Nine point of samples were observed under UV light and with I2. Nine components were obtained by silica column chromotography, different ratio of petroleum ether and ethyl acetate as eluent, yield rate of different component was calculated after condensing and drying solvent, and the antifungal activity was tested. The yield rate of component VI was higher, that is24.76%, and its inhibition rate on the tested plant pathogen was highest, showed that the main part of antifungal substance was in it. The232mg pure sample of component Ⅵ-1was obtained by sephadex LH20column chromotography with MeOH and water as eluent. The purity of component Ⅵ-1was above95%after HPLC analysis. After High resolution mass spectrometry determination, the deduced formula for this coumpound was C28H32O8. The mass spectrum was same as bislongiquinolide in NIST, the abundant was95%.
引文
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