瘤胃微生物Real Time PCR定量方法的建立及其应用
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摘要
本研究分别以产甲烷菌、原虫和瘤胃细菌为例,根据其16S/18S rDNA序列、ITS序列和特有蛋白的基因序列,利用Real Time PCR技术,分别在种和类的水平上,建立了对瘤胃微生物绝对定量与相对定量的分子生物学方法,为瘤胃微生物的研究提供了一种快速、准确的方法。同时,应用此方法研究了植物油对瘤胃中产甲烷菌数量、氢化细菌数量及原虫数量的影响,探讨了植物油降低瘤胃微生物甲烷产量的途径。
DNA的提取采用珠磨——酚氯仿法。提取的瘤胃微生物总DNA在20Kb以上,OD_(260)nm/OD_(280nm)均在1.7～1.9,提取效率达87.64%。利用真细菌、古细菌和真菌16S/18S rDNA的通用引物从纯化后瘤胃微生物总DNA中成功扩增出目标条带,表明珠磨式机械破碎法能充分破碎包括细菌、真菌、原虫在内的各种瘤胃微生物,并满足后续实验的要求。
利用Acc Ⅰ、EcoR Ⅰ、Pst Ⅰ、Pvu Ⅱ、Sac Ⅰ、Sma Ⅰ等6种限制性内切酶将基因组酶切,通过Southern杂交确定M.formicicum的16S rDNA序列在基因组中有2个拷贝。设计了M.formicicum种专一探针,通过在GeneBank中用Blast进行比对,证实该探针特异性很高。对M.formicicum的Real Time PCR反应条件进行了优化,其最佳体系为:引物和探针的浓度分别为0.4μmol/L、0.2μmol/L,MgCl_2 4mmol/L,模板浓度为126ng。检测灵敏度可达到30拷贝/25μL体系。M.formicicum采用Real Time PCR方法测得的数量低于采用传统最大或然数法测得的数量,但两者之间具有很高的相关性(r=0.957,P<0.01),说明采用Real Time PCR的方法能够如实的反映瘤胃微生物数量的变化。
以ITS为靶序列,采用SYBR Green Ⅰ荧光染料Real Time PCR法成功对M.mazei进行了定量检测,有效检测灵敏度可达300拷贝/25μL体系。同时,以微生物合成甲烷过程中的关键酶mcrA基因为靶基因,采用SYBR Green Ⅰ荧光染料Real Time PCR法,建立了瘤胃总产甲烷菌相对定量方法。利用上述方法,分别对肉牛、奶牛瘤胃中的M.formicicum、总产甲烷菌进行了检测,结果表明所测得的肉牛瘤胃中的M.formicicum、总产甲烷菌的数量远高于奶牛,这可能与肉牛日粮中粗料比例较高有关。同时,采用SYBR Green Ⅰ荧光染料Real Time PCR法成功对B.fibrivolen和R.ablus进行了定量检测。
日粮中添加4%棉籽油和4%豆油均降低人工瘤胃中微生物的甲烷产量,但二者之间无显著差异。不同时间段内,甲烷产量降低的幅度不同,其中采食后2～4h内甲烷产量降低的幅度最大,与对照组相比,分别降低25.52%和24.26%(P<0.05),4～6h次之,6～8h变化最小。添加4%棉籽油和4%豆油,使人工瘤胃中的M.formicium、总产甲烷菌、Ciliate protozoa、B.fibrivolen和R.ablus的数量均减少。与对照组相比,添加4%棉籽油组和添加4%豆油组,7d中部采样M.formicium、总产甲烷菌、Ciliate protozoa、B.fibrivolen和R.ablus的数量分别减少了100%、100%,91.40%、82.57%,87.50%、96.87%,6.05%、12.94%,71.54%、89.43%,在底部采样中,分别降低了100%、100%,36.49%、40.54%,61.98%、67.55%,11.94%、14.74%,54.62%、43.10%。上述结果显示,植物油主要是通过杀灭瘤胃原虫,从而减少产甲烷菌数量以降低甲烷的生成。
A perfect quantitative method of rumen microorganisms was developed by real time PCR as the example of methanogens, protozoa and rumen bacteria in this paper, on the sequence of 16S /18S rDNA, ITS and the genes of own protein basis. And the detecting and quantitative reaction system of methanogens was established from the angles of species and genera, absolute and relative quantification, known and unknown copy numbers. Furthermore, the regression relation was developed on comparing the method of real time PCR with the method of most probable number. At the same time, the effect of plant oil on the population of methanogens, able-hydrogenated bacteria, ciliate protozoa and methane production was studied by real time PCR, and the approach of plant oil reducing methane production of rumen microorganisms was discussed.The method of DNA extraction by a bead-beating technique and phenol-CHCl_3 was used. The DNA extracted was longer than 20kb, OD_(260nm)/OD_(280nm) of DNA was 1.7-1.9, and the efficiency of the DNA extraction was 87.64%. we got the target 16S rDNA sequences of the PCR-amplification by primers of bacteria, archaea and fungi. This showed that the method of DNA extraction in this paper could well break the cells of rumen microorganisms including bacteria, fungi and protozoa into pieces and could meet needs for other subsequent studies.It was determined by the digestion of genomic DNA with six different restriction endonucleases: AccⅠ, EcoRⅠ, PstⅠ, PvuⅡ, Sacland SmaⅠ and southern hybridization that the copy number of M.formicicum'sl6S rDNA was two. A special probe was designed and it was approved that exclusive sequences was own by aligning in GeneBank. We optimized the reaction condition of M. formicicum. When the concentrations of its primers and probe were 0.4μmol/L, 0.2μmol/L respectively and the concentration of MgCl_2 and templates were 4mmol/L and 1260ng respectively, the real time PCR reaction system of M. formicicum was best. The lowest copy number of M. formicicum which could be detected was 30 in 25μL reaction system. The population of M. formicicum by real time PCR was lower than the population of M. formicicum by the method of most probable number, and the positive correlation between them was very high(r=0.957. P<0.01). This showed the quantitative method by real time PCR could reflect the variation of rumen microorganism's population truly.We successfully detected M. mazei by real time PCR with SYBR GreenI when the sequence of ITS was used as a target sequence, and the lowest copy number of M. formicicum which could be well and truly detected was 300 in 25μL reaction system. At the same time, we successfully developed the method of methanogens' relative quantification by real time PCR with SYBR GreenI when the gene of mcrA was used as a target sequence. Furthermore, it showed that the population of M. formicicum and methanogens from rumen fluid of steers was more than those from rumen fluid of cows by those detection methods, it maybe relative to the diets of steers in which the ratio between concentration and forage was high. We also successfully detected B.fibrivolen and R.ablus by real time PCR with SYBR
DNA的提取采用珠磨——酚氯仿法。提取的瘤胃微生物总DNA在20Kb以上,OD_(260)nm/OD_(280nm)均在1.7～1.9,提取效率达87.64%。利用真细菌、古细菌和真菌16S/18S rDNA的通用引物从纯化后瘤胃微生物总DNA中成功扩增出目标条带,表明珠磨式机械破碎法能充分破碎包括细菌、真菌、原虫在内的各种瘤胃微生物,并满足后续实验的要求。
利用Acc Ⅰ、EcoR Ⅰ、Pst Ⅰ、Pvu Ⅱ、Sac Ⅰ、Sma Ⅰ等6种限制性内切酶将基因组酶切,通过Southern杂交确定M.formicicum的16S rDNA序列在基因组中有2个拷贝。设计了M.formicicum种专一探针,通过在GeneBank中用Blast进行比对,证实该探针特异性很高。对M.formicicum的Real Time PCR反应条件进行了优化,其最佳体系为:引物和探针的浓度分别为0.4μmol/L、0.2μmol/L,MgCl_2 4mmol/L,模板浓度为126ng。检测灵敏度可达到30拷贝/25μL体系。M.formicicum采用Real Time PCR方法测得的数量低于采用传统最大或然数法测得的数量,但两者之间具有很高的相关性(r=0.957,P<0.01),说明采用Real Time PCR的方法能够如实的反映瘤胃微生物数量的变化。
以ITS为靶序列,采用SYBR Green Ⅰ荧光染料Real Time PCR法成功对M.mazei进行了定量检测,有效检测灵敏度可达300拷贝/25μL体系。同时,以微生物合成甲烷过程中的关键酶mcrA基因为靶基因,采用SYBR Green Ⅰ荧光染料Real Time PCR法,建立了瘤胃总产甲烷菌相对定量方法。利用上述方法,分别对肉牛、奶牛瘤胃中的M.formicicum、总产甲烷菌进行了检测,结果表明所测得的肉牛瘤胃中的M.formicicum、总产甲烷菌的数量远高于奶牛,这可能与肉牛日粮中粗料比例较高有关。同时,采用SYBR Green Ⅰ荧光染料Real Time PCR法成功对B.fibrivolen和R.ablus进行了定量检测。
日粮中添加4%棉籽油和4%豆油均降低人工瘤胃中微生物的甲烷产量,但二者之间无显著差异。不同时间段内,甲烷产量降低的幅度不同,其中采食后2～4h内甲烷产量降低的幅度最大,与对照组相比,分别降低25.52%和24.26%(P<0.05),4～6h次之,6～8h变化最小。添加4%棉籽油和4%豆油,使人工瘤胃中的M.formicium、总产甲烷菌、Ciliate protozoa、B.fibrivolen和R.ablus的数量均减少。与对照组相比,添加4%棉籽油组和添加4%豆油组,7d中部采样M.formicium、总产甲烷菌、Ciliate protozoa、B.fibrivolen和R.ablus的数量分别减少了100%、100%,91.40%、82.57%,87.50%、96.87%,6.05%、12.94%,71.54%、89.43%,在底部采样中,分别降低了100%、100%,36.49%、40.54%,61.98%、67.55%,11.94%、14.74%,54.62%、43.10%。上述结果显示,植物油主要是通过杀灭瘤胃原虫,从而减少产甲烷菌数量以降低甲烷的生成。
A perfect quantitative method of rumen microorganisms was developed by real time PCR as the example of methanogens, protozoa and rumen bacteria in this paper, on the sequence of 16S /18S rDNA, ITS and the genes of own protein basis. And the detecting and quantitative reaction system of methanogens was established from the angles of species and genera, absolute and relative quantification, known and unknown copy numbers. Furthermore, the regression relation was developed on comparing the method of real time PCR with the method of most probable number. At the same time, the effect of plant oil on the population of methanogens, able-hydrogenated bacteria, ciliate protozoa and methane production was studied by real time PCR, and the approach of plant oil reducing methane production of rumen microorganisms was discussed.The method of DNA extraction by a bead-beating technique and phenol-CHCl_3 was used. The DNA extracted was longer than 20kb, OD_(260nm)/OD_(280nm) of DNA was 1.7-1.9, and the efficiency of the DNA extraction was 87.64%. we got the target 16S rDNA sequences of the PCR-amplification by primers of bacteria, archaea and fungi. This showed that the method of DNA extraction in this paper could well break the cells of rumen microorganisms including bacteria, fungi and protozoa into pieces and could meet needs for other subsequent studies.It was determined by the digestion of genomic DNA with six different restriction endonucleases: AccⅠ, EcoRⅠ, PstⅠ, PvuⅡ, Sacland SmaⅠ and southern hybridization that the copy number of M.formicicum'sl6S rDNA was two. A special probe was designed and it was approved that exclusive sequences was own by aligning in GeneBank. We optimized the reaction condition of M. formicicum. When the concentrations of its primers and probe were 0.4μmol/L, 0.2μmol/L respectively and the concentration of MgCl_2 and templates were 4mmol/L and 1260ng respectively, the real time PCR reaction system of M. formicicum was best. The lowest copy number of M. formicicum which could be detected was 30 in 25μL reaction system. The population of M. formicicum by real time PCR was lower than the population of M. formicicum by the method of most probable number, and the positive correlation between them was very high(r=0.957. P<0.01). This showed the quantitative method by real time PCR could reflect the variation of rumen microorganism's population truly.We successfully detected M. mazei by real time PCR with SYBR GreenI when the sequence of ITS was used as a target sequence, and the lowest copy number of M. formicicum which could be well and truly detected was 300 in 25μL reaction system. At the same time, we successfully developed the method of methanogens' relative quantification by real time PCR with SYBR GreenI when the gene of mcrA was used as a target sequence. Furthermore, it showed that the population of M. formicicum and methanogens from rumen fluid of steers was more than those from rumen fluid of cows by those detection methods, it maybe relative to the diets of steers in which the ratio between concentration and forage was high. We also successfully detected B.fibrivolen and R.ablus by real time PCR with SYBR
引文
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