粘细菌的分离纯化和分类鉴定
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摘要
本实验以采集自越南以及我国的云南、河北、陕西各地的土样和各种树皮、腐木作为实验材料，利用兔、羊粪球和滤纸作为基质诱导出多种颜色丰富、形态多样的粘细菌子实体，采用本实验室摸索出的较为有效的纯化粘细菌的方法，在实验器材上用24孔细胞培养板代替传统的平皿，有效地缩短了粘细菌的纯化周期，提高了分离纯化的效率。从越南、云南、河北、陕西各地的94份土壤样品和9份树皮、腐木样品中，分离得到259株粘细菌，并从中纯化出140株。
根据菌株的特征性子实体形态和其营养细胞、粘孢子形态，以《Bergey's manualof determinative bacteriology》第九版为依据，将分离纯化出的菌株初步鉴定到属，这259株粘细菌菌株经过初步的分类鉴定，分属于9个属，即粘球菌属(Myxococcus)139株、珊瑚球菌属(Corallococcus)20株、囊球菌属(Angiococcus)18株、原囊菌属(Archangium)21株、孢囊杆菌属(Cystobacter)5株、标桩菌属(Stigmatella)36株、单囊菌属(Haploangium)9株、小囊菌属(Nannocystis)3株，和多囊菌属(Polyangium)8株。对纯化菌株的液体培养特征进行了描述，并首次依据初步分类鉴定结果探索了液体特征与分类地位之间的关系，即依据营养细胞和粘孢子形态将粘细菌划分成的两大类型中，第一类型(包括粘球菌属等7个属)的菌株在液体培养基中通常形成片状或絮状，而第二类型(包括单囊菌属等5个属)的菌株则主要呈现球形。
比较讨论了丙酮提取法、碱裂解法和创新的NaOH-CTAB法提取粘细菌总DNA的效果，较之丙酮提取法和碱裂解法，NaOH-CTAB法可以更加彻底、快速地破坏粘细菌细胞壁，同时可以较彻底去除粘细菌丰富的胞外多糖，从而得到高质量的粘细菌总DNA，使粘细菌的分子生物学研究更为简便。
选取具有抑制肿瘤活性的粘细菌菌株6株(菌株编号分别为：YN34、YN65、YN76、YN112、BD85、BD105)，根据形态特征将其归为粘球菌属(Myxococcus)。应用NaOH-CTAB法提取这6株粘细菌菌株的总DNA，进行16S rDNA序列测定，从分子水平初步确定其分类地位。将其16S rDNA基因序列与GenBank中所有已
河北大学理学硕士学位论文
经测定的原核生物的165 rDNA序列进行比较，构建了粘球菌属你为优ococcu:)
的系统发育进化树，确定其系统发育地位，并进行了生理生化反应实验以及总DNA
的G+Cmol%测定。结果表明，这6株菌的G+Cmol%均比较高，在65十69%之间;
它们均属于粘球菌属。为优口coccus)，菌株YN34与菌株YNll2和BDzOS关系均较
近，相似性均为10既，与它们关系最近的为橙色粘球菌以办优ococcus fulvus)
AJ233919，相似性为99.78%;YN65与大抱粘球菌。心优口coecus macro明orus)
AYO72738关系最近，相似性为99.57%;YN76与菌株YN34、YNI 12、和BD105关系
均较近，相似性均为99.86%，与橙色粘球菌。为优口eoecusfulvus)AJ233919，相似
性为99.64%;BD85与黄色粘球菌帆乃优口coceus xanthus)M34114关系最近，相
似性为100%。
生理生化实验结果与165 rDNA序列分析结果基本一致，证明了生理生化实验
对于粘细菌分类的重要作用。另外，在一些生理生化反应上，二者又有较大的差
异，体现出了它与分子分类在内的其他分类方法相结合，以共同准确地确定粘细
菌的真实分类地位的必要性。
The soil and bark samples collected from Yunnan, Hebei and Shanxi provinces of China, and also from Vietnam, were used as the materials in this experiment. And dung pellets of rabbits or goats that had been autoclaved can be used to induce many kinds of fruiting bodies of the Myxobacteria in different colors, sizes, and shapes. We isolated and purified Myxobacteria strains with the improved method which was found by our laboratory. The using of tissue culture plates instead of traditional plates accelerated the purification process and enhanceed the purification efficiency. From 94 soil samples and 9 bark samples in these areas, 259 Myxobacteria strains were isolated and 140 strains of them were purified.
These 259 Myxobacteria strains were primarily identified in genus or species level by their fruiting bodies, myxospores and vegetative cells on the base of Sergey's manual of determinative bacteriology and they belonged to 9 genera(Myxococcus, Corallococcm. Angiococcus. Archangium, Cystobacter. Stigmatella . Hapoangium. Nannocystis. Polyangium) of Myxococcales. The characteristic in liquid culture of the purified strains were described, and the relation between the characteristic and the taxonomic traits was researched for the first time. The two groups of Myxobacteria differentiated according to the shape character of the myxospores and vegetative cells had different characteristic in liquid culture. The strains belonged to the first group including Myxococcus, Corallococcus, Angiococcus, Archangium, Cystobacter, Melittangium and Stigmatella were usual pieces or floe in liquid culture, while strains of the other group including Hapoangium, Nannocystis, Polyangium, Sorangium and Chondromyces were spheriform
mostly.
The effect of three methods for extracting total DNA of Myxobacteria which were Acetone isolation method .Alkaline lysis method and innovative NaOH-CTAB method was compared. The NaOH-CTAB method can destroy cell wall of Myxobacteria more drastically and wipe off extracellular amylose more effectively than by Acetone
isolation method and Alkaline lysis method which offer total DNA of high quality for the study of molecular biology of Myxobacetia.
Six strains(serial numbers were YN34, YN65, YN76, YN112, BD85, BD105) which had the inhibitory activity to the tumour belonged to Myxococcus according to their shapes and their total DNA were extracted by the NaOH-CTAB method. The 16S rDNA sequencing analyses of them was carried out. Complete sequences of them were obtained and phylogenesis analyses were carried out of Myxococcus, their phylogenetic position was determined. We did also G+Cmol%, physiological and biochemical characteristics tests. The results show that the 6 strains should be clustered into the genus Myxococcus; YN34, YN112 and BD105 had close relationship and their sequence similarity were 100%, the most closed strain was Myxococcus fulvus AJ233919; YN65 was similar to Myxococcus macrosporus; YN76 was closed to YN34, YN112 and BD105 and their sequence comparability were 99.86%; BD85 similar to Myxococcus xanthus. G+CmoI% of the 6 strains were in the range of 65% to 69%.
The 16S rDNA sequences results were almost identical to the physiological and biochemical results, this conformed the importance of molecular, physiological and biochemical function in the taxonomy of Myxobacteria . While the physiological and biochemical function and other taxology methods were need to combined for the exist of some differences between them.
根据菌株的特征性子实体形态和其营养细胞、粘孢子形态，以《Bergey's manualof determinative bacteriology》第九版为依据，将分离纯化出的菌株初步鉴定到属，这259株粘细菌菌株经过初步的分类鉴定，分属于9个属，即粘球菌属(Myxococcus)139株、珊瑚球菌属(Corallococcus)20株、囊球菌属(Angiococcus)18株、原囊菌属(Archangium)21株、孢囊杆菌属(Cystobacter)5株、标桩菌属(Stigmatella)36株、单囊菌属(Haploangium)9株、小囊菌属(Nannocystis)3株，和多囊菌属(Polyangium)8株。对纯化菌株的液体培养特征进行了描述，并首次依据初步分类鉴定结果探索了液体特征与分类地位之间的关系，即依据营养细胞和粘孢子形态将粘细菌划分成的两大类型中，第一类型(包括粘球菌属等7个属)的菌株在液体培养基中通常形成片状或絮状，而第二类型(包括单囊菌属等5个属)的菌株则主要呈现球形。
比较讨论了丙酮提取法、碱裂解法和创新的NaOH-CTAB法提取粘细菌总DNA的效果，较之丙酮提取法和碱裂解法，NaOH-CTAB法可以更加彻底、快速地破坏粘细菌细胞壁，同时可以较彻底去除粘细菌丰富的胞外多糖，从而得到高质量的粘细菌总DNA，使粘细菌的分子生物学研究更为简便。
选取具有抑制肿瘤活性的粘细菌菌株6株(菌株编号分别为：YN34、YN65、YN76、YN112、BD85、BD105)，根据形态特征将其归为粘球菌属(Myxococcus)。应用NaOH-CTAB法提取这6株粘细菌菌株的总DNA，进行16S rDNA序列测定，从分子水平初步确定其分类地位。将其16S rDNA基因序列与GenBank中所有已
河北大学理学硕士学位论文
经测定的原核生物的165 rDNA序列进行比较，构建了粘球菌属你为优ococcu:)
的系统发育进化树，确定其系统发育地位，并进行了生理生化反应实验以及总DNA
的G+Cmol%测定。结果表明，这6株菌的G+Cmol%均比较高，在65十69%之间;
它们均属于粘球菌属。为优口coccus)，菌株YN34与菌株YNll2和BDzOS关系均较
近，相似性均为10既，与它们关系最近的为橙色粘球菌以办优ococcus fulvus)
AJ233919，相似性为99.78%;YN65与大抱粘球菌。心优口coecus macro明orus)
AYO72738关系最近，相似性为99.57%;YN76与菌株YN34、YNI 12、和BD105关系
均较近，相似性均为99.86%，与橙色粘球菌。为优口eoecusfulvus)AJ233919，相似
性为99.64%;BD85与黄色粘球菌帆乃优口coceus xanthus)M34114关系最近，相
似性为100%。
生理生化实验结果与165 rDNA序列分析结果基本一致，证明了生理生化实验
对于粘细菌分类的重要作用。另外，在一些生理生化反应上，二者又有较大的差
异，体现出了它与分子分类在内的其他分类方法相结合，以共同准确地确定粘细
菌的真实分类地位的必要性。
The soil and bark samples collected from Yunnan, Hebei and Shanxi provinces of China, and also from Vietnam, were used as the materials in this experiment. And dung pellets of rabbits or goats that had been autoclaved can be used to induce many kinds of fruiting bodies of the Myxobacteria in different colors, sizes, and shapes. We isolated and purified Myxobacteria strains with the improved method which was found by our laboratory. The using of tissue culture plates instead of traditional plates accelerated the purification process and enhanceed the purification efficiency. From 94 soil samples and 9 bark samples in these areas, 259 Myxobacteria strains were isolated and 140 strains of them were purified.
These 259 Myxobacteria strains were primarily identified in genus or species level by their fruiting bodies, myxospores and vegetative cells on the base of Sergey's manual of determinative bacteriology and they belonged to 9 genera(Myxococcus, Corallococcm. Angiococcus. Archangium, Cystobacter. Stigmatella . Hapoangium. Nannocystis. Polyangium) of Myxococcales. The characteristic in liquid culture of the purified strains were described, and the relation between the characteristic and the taxonomic traits was researched for the first time. The two groups of Myxobacteria differentiated according to the shape character of the myxospores and vegetative cells had different characteristic in liquid culture. The strains belonged to the first group including Myxococcus, Corallococcus, Angiococcus, Archangium, Cystobacter, Melittangium and Stigmatella were usual pieces or floe in liquid culture, while strains of the other group including Hapoangium, Nannocystis, Polyangium, Sorangium and Chondromyces were spheriform
mostly.
The effect of three methods for extracting total DNA of Myxobacteria which were Acetone isolation method .Alkaline lysis method and innovative NaOH-CTAB method was compared. The NaOH-CTAB method can destroy cell wall of Myxobacteria more drastically and wipe off extracellular amylose more effectively than by Acetone
isolation method and Alkaline lysis method which offer total DNA of high quality for the study of molecular biology of Myxobacetia.
Six strains(serial numbers were YN34, YN65, YN76, YN112, BD85, BD105) which had the inhibitory activity to the tumour belonged to Myxococcus according to their shapes and their total DNA were extracted by the NaOH-CTAB method. The 16S rDNA sequencing analyses of them was carried out. Complete sequences of them were obtained and phylogenesis analyses were carried out of Myxococcus, their phylogenetic position was determined. We did also G+Cmol%, physiological and biochemical characteristics tests. The results show that the 6 strains should be clustered into the genus Myxococcus; YN34, YN112 and BD105 had close relationship and their sequence similarity were 100%, the most closed strain was Myxococcus fulvus AJ233919; YN65 was similar to Myxococcus macrosporus; YN76 was closed to YN34, YN112 and BD105 and their sequence comparability were 99.86%; BD85 similar to Myxococcus xanthus. G+CmoI% of the 6 strains were in the range of 65% to 69%.
The 16S rDNA sequences results were almost identical to the physiological and biochemical results, this conformed the importance of molecular, physiological and biochemical function in the taxonomy of Myxobacteria . While the physiological and biochemical function and other taxology methods were need to combined for the exist of some differences between them.
引文
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