基于红树林土壤微生物资源研发的宏基因组学平台技术的建立与应用初探
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摘要
红树林土壤处于“海洋-陆地”界面潮间带环境,其生境的独特性决定了其中微生物的多样性及基因资源的珍稀性,深入开展红树林土壤微生物的研究将加快新型功能基因、新颖天然产物的发现进程。完全不依赖于平板分离的现代宏基因组学技术为充分探索不同自然生境中的微生物资源提供了有力手段。然而,将该技术实施于红树林土壤微生物的研究时,尚存在若干困难:①红树林土壤为高粘质、高有机质含量类型土壤,提取DNA时存在大量腐殖酸及腐殖酸类等高分子量抑制性物质的共提取等问题,造成所得DNA品质低下,难于进行微生物多样性分析。②DNA提取过程中,红树林土壤中高含量的粘质极易吸附刚从微生物细胞裂解释放的大片段DNA,造成所得DNA提取物中大片段DNA所占比率低下,难以构建大片段宏基因组文库应用于特色微生物资源的“生物探矿”。针对上述难点,本论文系统开展了红树林土壤DNA提取缓冲液的改良与应用研究;红树林土壤大片段宏基因组文库构建方法的改良与应用研究;并针对现行环境宏基因组学技术所存在的“群落结构偏差”和“基因遗漏”等瓶颈,开展了不同裂解方式对红树林土壤不同类型土著微生物细胞裂解能力的研究;获得了如下主要研究结果与结论:
1.提取土壤DNA时,缓冲液中的NaCl是决定抑制性物质共提取量高低的关键因素之一,随NaCl浓度的升高,腐殖酸等抑制性物质的共提取量明显下降,而Na_3PO_4对抑制性物质共提取量的影响却呈现完全相反的规律;PVP在土壤微生物细胞裂解前及裂解过程中使用均增加抑制性物质的共提取量,裂解后使用反而能提高DNA提取物的品质;PVPP在土壤微生物细胞裂解前及裂解过程中使用均减少抑制性物质的共提取量,裂解后使用反而无效;CTAB在土壤微生物细胞裂解前、裂解过程中及裂解后使用均能提高DNA初提物的品质;随缓冲液中SDS浓度的升高和裂解时间的延长,抑制性物质的共提取量明显增加:随缓冲液中CTAB浓度的升高,抑制性物质的共提取量逐渐减少,随裂解时间的延长,抑制性物质的共提取量表现为先增加后减少的规律;CTAB在缓冲液中使用具有裂解微生物细胞和去除抑制性物质的双重作用,去除抑制性物质的作用占主导地位;PVP、PVPP及CTAB在不同浓度及不同作用方式下使用均不改变环境样品的细菌群落结构;提取缓冲液含1.5mol.L~(-1)NaCl,2%CTAB及2%PVPP时,所得红树林土壤DNA初提物不经纯化即可成功进行PCR扩增反应。
2.运用本研究改良后的原位裂解提取法能有效提高大片段DNA在总土壤DNA中的比率,该方法与电洗脱纯化法联用能高效构建红树林土壤大片段宏基因组文库。本论文所得4个季节红树林土壤宏基因组文库,含9570个cosmid克隆子,每个克隆子的平均插入片段均大于35kbp。运用功能筛选方法,从该文库中得到1株具有四环素抗性的克隆子,1株产褐色色素的克隆子和1株具有淀粉酶活性的克隆子。
3.通过古生菌、细菌、真菌及放线细菌特异性引物对不同裂解方法所得红树林土壤DNA进行PCR反应,结果表明:SDS、溶菌酶、液氮冻融、玻璃珠剧烈振荡、超声波、微波等6种常规裂解方式单独应用均能裂解红树林土壤土著细菌和古生菌,均不能裂解红树林土壤土著放线细菌和真菌;SDS、溶菌酶及玻璃珠剧烈振荡等3种方法联用时能裂解红树林土壤土著真菌;SDS、溶菌酶、玻璃珠剧烈振荡及微波等4种方法联用时能裂解红树林土壤土著放线细菌。
4.运用DGGE方法比较上述6种裂解方式捕捉红树林土壤土著细菌群落结构信息的能力,结果显示:没有1种裂解方式能获得完整的细菌群落结构信息;超声波、微波裂解法所得红树林土壤细菌群落结构与溶菌酶裂解法所得结果相似;其它4种裂解法所得红树林土壤细菌群落结构各不相同,每种裂解方法均含有其它方法所遗漏的物种信息。
5.红树林土壤微生物胞外DNA包含细菌和古生菌的基因组信息,不包含放线细菌及真菌的基因组信息;同种红树植物-白骨壤根际土壤细菌群落受土壤深度及季节变化因素影响小。
红树林土壤微生物宏基因组学研究平台技术的建立,不仅为潮间带特色微生物的生态学基础研究、生物技术应用研究提供了有力的技术保障,而且为其它生境宏基因组学领域的研究提供了有益借鉴。
Microbes inhabiting in special intertidal mangrove soil niches possess the enormous genetic diversity and immense potential to produce new secondary metabolites. Paying attention to the research mangrove soil microbes would accelerate the rate of discovering novel bioactive molecules. Culture-independent metagenomic technology provides a powerful tool to explore microbial resources with high throughput in diverse niches, however, some hurdles have not yet overcomed when it applied to mangrove soil niches. ( i) Mangrove soil is high in clay and rich in organic matter, the abundant high molecular weight inhibitors such as humic acid and humic compounds in soil samples are difficult to remove, and are always co-extracted with soil DNA, subsequent molecular diversity analysis would be hindered because of the poor quality of the isolated DNA. (ii) In the lysis step, DNA liberated from soil microbes, especially the large fragment DNA, was likely to adsorb to the soil clay. Therefore, the total extracts with low percentage of large fragment DNA failed to construct large insert environmental libraries for biotechnology use. Towards overcoming these hurdles, the method of extraction of mangrove soil DNA for microbial diversity research and the method of construction of large fragment metagenome library of mangrove soil were studied in this paper. The results were as follows:
1. NaCl posed significance effect on yield of coextracted humic compounds (YCHC). YCHC decreased when the concentration of NaCl in the extraction buffer increased. Whereas, the effect of Na_3PO_4 on YCHC was just on the opposite; when polyvinylpyrrolidone (PVP) was used pre-lysis or in lysis, YCHC increased, whereas, when PVP was used after lysis, the quality of crude extracts was improved; when polyvinylpolypyrrolidone( PVPP) was used pre- lysis or in lysis, YCHC decreased, whereas, when PVPP wae used after lysis, it has no positive effect on the quality of crude extracts; Whenever CTAB was used pre lysis, in lysis or after lysis,, the quality of crude extracts was improved; When the concentration of SDS in the extraction buffer increased or the lysis time lasted more longer, the YCHC increased; When the concentration of CTAB in the extraction buffer increased, the YCHC decreased; The relationship between YCHC and lysis time with CTAB was as follows, the YCHC increased in the first two hours, then decreased gradually; The addition of CTAB in the extraction buffer not only resulted in lysing microbial cells but also removing the humic compounds, the latter one was the main function; Whenever PVP, PVPP, CTAB were used pre lysis, in lysis or after lysis, it does not resulted in the loss of microbial diversity; The quality of mangrove soil DNA without further purification was adequate for PCR when NaCl[1.5mol.L~(-1)], PVPP[2%] and CTAB[2%] introduced in the extraction buffer.
2. Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significantly increased, and the large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. This library produced 9570 cosmid clone, the average insert size for this library was larger than 35kbp. At least 335Mbp valuable genetic information of mangrove soil microbes was encompassed in this culturing-independent library. A clone containing anti-tetracylin activity, a clone producing brown pigment and a clone containing amylase activity were detected by function- driven screening method.
3. Experiments were designed to investigate the capability of capturing the indigenous microbial genome of SDS, lysomyze, shaking with bead beating, sonication, microwave and froze-thawn cycles in this study. Results showed, (i) Each methods could lyse the indigenous bacteria and archaea of mangrove soil, but could not lyse the indigenous actinobacteria and fungi. ( ii ) Indigenous fungi lysed when applying the the combination of SDS, lysomyze and shaking with bead beating. (iii) Indigenous actinobacteria lysed when applying the the combination of SDS, lysomyze, shaking with bead beating and microwave.
4. DGGE was used to compare the mangrove soil community produced by six methods respectively. Results showed, none method could produce whole bacterial community, the DGGE profile produced by soniction, microwave and lysomyze were similar, the DGGE profile produced by SDS, shaking with bead beating, froze-thawn cycles and froze-thawn cycles contained special band respectively.
5. The mangrove soil microbial extracellular DNA diversity was evaluated by PCR and the seasonal variation of bacterial diversity in the Avicennia marina rhizosphere was analyzed by DGGE. Results showed, DNA derived from bacteria and archaea were involved in extracellular DNA, but DNA derived from actinobacteria and fungi were excluded. The bacterial communities in the Avicennia marina rhizosphere of different depth in different seasons were similar.
In summary, Establishment of metagenomic technology platform allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.
1.提取土壤DNA时,缓冲液中的NaCl是决定抑制性物质共提取量高低的关键因素之一,随NaCl浓度的升高,腐殖酸等抑制性物质的共提取量明显下降,而Na_3PO_4对抑制性物质共提取量的影响却呈现完全相反的规律;PVP在土壤微生物细胞裂解前及裂解过程中使用均增加抑制性物质的共提取量,裂解后使用反而能提高DNA提取物的品质;PVPP在土壤微生物细胞裂解前及裂解过程中使用均减少抑制性物质的共提取量,裂解后使用反而无效;CTAB在土壤微生物细胞裂解前、裂解过程中及裂解后使用均能提高DNA初提物的品质;随缓冲液中SDS浓度的升高和裂解时间的延长,抑制性物质的共提取量明显增加:随缓冲液中CTAB浓度的升高,抑制性物质的共提取量逐渐减少,随裂解时间的延长,抑制性物质的共提取量表现为先增加后减少的规律;CTAB在缓冲液中使用具有裂解微生物细胞和去除抑制性物质的双重作用,去除抑制性物质的作用占主导地位;PVP、PVPP及CTAB在不同浓度及不同作用方式下使用均不改变环境样品的细菌群落结构;提取缓冲液含1.5mol.L~(-1)NaCl,2%CTAB及2%PVPP时,所得红树林土壤DNA初提物不经纯化即可成功进行PCR扩增反应。
2.运用本研究改良后的原位裂解提取法能有效提高大片段DNA在总土壤DNA中的比率,该方法与电洗脱纯化法联用能高效构建红树林土壤大片段宏基因组文库。本论文所得4个季节红树林土壤宏基因组文库,含9570个cosmid克隆子,每个克隆子的平均插入片段均大于35kbp。运用功能筛选方法,从该文库中得到1株具有四环素抗性的克隆子,1株产褐色色素的克隆子和1株具有淀粉酶活性的克隆子。
3.通过古生菌、细菌、真菌及放线细菌特异性引物对不同裂解方法所得红树林土壤DNA进行PCR反应,结果表明:SDS、溶菌酶、液氮冻融、玻璃珠剧烈振荡、超声波、微波等6种常规裂解方式单独应用均能裂解红树林土壤土著细菌和古生菌,均不能裂解红树林土壤土著放线细菌和真菌;SDS、溶菌酶及玻璃珠剧烈振荡等3种方法联用时能裂解红树林土壤土著真菌;SDS、溶菌酶、玻璃珠剧烈振荡及微波等4种方法联用时能裂解红树林土壤土著放线细菌。
4.运用DGGE方法比较上述6种裂解方式捕捉红树林土壤土著细菌群落结构信息的能力,结果显示:没有1种裂解方式能获得完整的细菌群落结构信息;超声波、微波裂解法所得红树林土壤细菌群落结构与溶菌酶裂解法所得结果相似;其它4种裂解法所得红树林土壤细菌群落结构各不相同,每种裂解方法均含有其它方法所遗漏的物种信息。
5.红树林土壤微生物胞外DNA包含细菌和古生菌的基因组信息,不包含放线细菌及真菌的基因组信息;同种红树植物-白骨壤根际土壤细菌群落受土壤深度及季节变化因素影响小。
红树林土壤微生物宏基因组学研究平台技术的建立,不仅为潮间带特色微生物的生态学基础研究、生物技术应用研究提供了有力的技术保障,而且为其它生境宏基因组学领域的研究提供了有益借鉴。
Microbes inhabiting in special intertidal mangrove soil niches possess the enormous genetic diversity and immense potential to produce new secondary metabolites. Paying attention to the research mangrove soil microbes would accelerate the rate of discovering novel bioactive molecules. Culture-independent metagenomic technology provides a powerful tool to explore microbial resources with high throughput in diverse niches, however, some hurdles have not yet overcomed when it applied to mangrove soil niches. ( i) Mangrove soil is high in clay and rich in organic matter, the abundant high molecular weight inhibitors such as humic acid and humic compounds in soil samples are difficult to remove, and are always co-extracted with soil DNA, subsequent molecular diversity analysis would be hindered because of the poor quality of the isolated DNA. (ii) In the lysis step, DNA liberated from soil microbes, especially the large fragment DNA, was likely to adsorb to the soil clay. Therefore, the total extracts with low percentage of large fragment DNA failed to construct large insert environmental libraries for biotechnology use. Towards overcoming these hurdles, the method of extraction of mangrove soil DNA for microbial diversity research and the method of construction of large fragment metagenome library of mangrove soil were studied in this paper. The results were as follows:
1. NaCl posed significance effect on yield of coextracted humic compounds (YCHC). YCHC decreased when the concentration of NaCl in the extraction buffer increased. Whereas, the effect of Na_3PO_4 on YCHC was just on the opposite; when polyvinylpyrrolidone (PVP) was used pre-lysis or in lysis, YCHC increased, whereas, when PVP was used after lysis, the quality of crude extracts was improved; when polyvinylpolypyrrolidone( PVPP) was used pre- lysis or in lysis, YCHC decreased, whereas, when PVPP wae used after lysis, it has no positive effect on the quality of crude extracts; Whenever CTAB was used pre lysis, in lysis or after lysis,, the quality of crude extracts was improved; When the concentration of SDS in the extraction buffer increased or the lysis time lasted more longer, the YCHC increased; When the concentration of CTAB in the extraction buffer increased, the YCHC decreased; The relationship between YCHC and lysis time with CTAB was as follows, the YCHC increased in the first two hours, then decreased gradually; The addition of CTAB in the extraction buffer not only resulted in lysing microbial cells but also removing the humic compounds, the latter one was the main function; Whenever PVP, PVPP, CTAB were used pre lysis, in lysis or after lysis, it does not resulted in the loss of microbial diversity; The quality of mangrove soil DNA without further purification was adequate for PCR when NaCl[1.5mol.L~(-1)], PVPP[2%] and CTAB[2%] introduced in the extraction buffer.
2. Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significantly increased, and the large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. This library produced 9570 cosmid clone, the average insert size for this library was larger than 35kbp. At least 335Mbp valuable genetic information of mangrove soil microbes was encompassed in this culturing-independent library. A clone containing anti-tetracylin activity, a clone producing brown pigment and a clone containing amylase activity were detected by function- driven screening method.
3. Experiments were designed to investigate the capability of capturing the indigenous microbial genome of SDS, lysomyze, shaking with bead beating, sonication, microwave and froze-thawn cycles in this study. Results showed, (i) Each methods could lyse the indigenous bacteria and archaea of mangrove soil, but could not lyse the indigenous actinobacteria and fungi. ( ii ) Indigenous fungi lysed when applying the the combination of SDS, lysomyze and shaking with bead beating. (iii) Indigenous actinobacteria lysed when applying the the combination of SDS, lysomyze, shaking with bead beating and microwave.
4. DGGE was used to compare the mangrove soil community produced by six methods respectively. Results showed, none method could produce whole bacterial community, the DGGE profile produced by soniction, microwave and lysomyze were similar, the DGGE profile produced by SDS, shaking with bead beating, froze-thawn cycles and froze-thawn cycles contained special band respectively.
5. The mangrove soil microbial extracellular DNA diversity was evaluated by PCR and the seasonal variation of bacterial diversity in the Avicennia marina rhizosphere was analyzed by DGGE. Results showed, DNA derived from bacteria and archaea were involved in extracellular DNA, but DNA derived from actinobacteria and fungi were excluded. The bacterial communities in the Avicennia marina rhizosphere of different depth in different seasons were similar.
In summary, Establishment of metagenomic technology platform allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.
引文
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