水稻土细菌和氨氧化细菌的RFLP分析
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摘要
细菌在生态系统的能量转化和食物链中既是初级生产者又是分解者,细菌的多样性在农业生态系统中又具有各种不同的功能。氮是植物生长所必需的营养元素,是土壤生产力的重要限制性因素。硝化作用中第一步反应NH_4~+一NO_2~-由氨氧化细菌(AOB)完成,是硝化过程的限速步骤。
水稻是人类赖以生存的粮食作物,水稻根际是一个特殊的生态环境,水稻根际微生物对于水稻的生产、土壤结构与肥力维持,以及在营养元素的流动过程中起着重要的作用。通过对水稻根际土细菌和氨氧化细菌多样性的研究,阐明根际土壤环境与细菌及氨氧化细菌群落结构之间的相互关系,可以加深人们对植物-土壤-微生物相互关系的理解和认识,对提高作物产量、保护环境等方面具有重要的意义。
本试验以陕西省武功县大田苗期水稻鲜土为研究对象,通过直接法提取根际水稻土和非根际水稻土微生物总DNA。采用细菌16S rDNA通用引物对(63F/1387R)和氨氧化细菌16S rDNA特异性引物(Eub338和Nso1225)分别对细菌及氨氧化细菌16S rDNA进行PCR扩增,将扩增片段与pMD19-T载体连接后转入大肠杆菌JM109中,通过蓝白斑筛选挑取阳性克隆子,分别建立水稻根际土和非根际土细菌、水稻根际土和非根际土氨氧化细菌等4个16S rDNA克隆文库。通过菌落PCR方法,从4个文库中分别随机挑取约150个阳性克隆,以pMD19-T载体通用引物M13重新扩增插入的16S rDNA片段,进行克隆文库的菌落PCR,将纯化后的菌落PCR产物分别用Hha I和Rsa I两种限制性内切酶水解,进行RFLP分型分析。
利用α多样性的测度分析RFLP的结果,聚类分析以细菌分布之间的Bray-Curtis相异性测度系数为距离指标,用非加权平均配对法(UPGA)在NTsys 2.10统计软件上进行数据处理。选取水稻根际土氨氧化细菌的优势克隆进行测序,所得的部分16S rDNA序列在NCBI上BLASTN比较,确定优势氨氧化细菌的种属;综合测序所得序列及NCBI上的相关序列,通过Cluster X及MEGA4.0构建进化树。
获得的主要试验结果如下:
(1)从水稻根际土中获得124个细菌的酶切类型,库容值为21.28%;从水稻非根际土中获得116个细菌的酶切类型,库容值为27.86%;从水稻根际土中获得110个氨氧化细菌的酶切类型,库容值为51.70%;从水稻非根际土中获得105个氨氧化细菌的酶切类型,库容值为52.26%。
(2)采用α多样性的测度对所得的细菌的RFLP分型数据进行分析,Shannon-Wiener(H′)多样性指数:水稻根际土为4.764,水稻非根际土为4.658;Margalef丰富度指数(d_(Ma)):水稻根际土为24.855,水稻非根际土为23.272;Pielou均匀度指数(J_(gi)):水稻根际土为0.988,水稻非根际土为0.980。其结果由大至小均为根际水稻土大于非根际水稻土。结果表明,水稻根际土中微生物物种丰富度较高,种类分布较均匀,构成功能体系较完善的微生物群落。
(3)采用α多样性的测度对所得的氨氧化细菌的RFLP分型数据进行分析,Gini(D_g)多样性指数:水稻根际土为0.984,水稻非根际土为0.986;均匀度指数(J_(gi)):水稻根际土为0.952,水稻非根际土为0.966;物种丰富度指数(d_(Ma)):水稻根际土为21.297,水稻非根际土为20.621;最大物种多样性指数(H_(max)):水稻根际土为4.701,水稻非根际土为4.654。
(4)根际水稻土优势氨氧化细菌G5、G13、G14、G26、G33、G42和G111的16S rDNA序列比对表明:G14和G111与亚硝化螺菌属(Nitrosospira sp.)同源性分别达到97%和93%; G33和G13与未培养亚硝化单胞菌属(Uncultured Nitrosomonadaceae bacterium)同源性分别达到98%和92%;G26和G42与未培养β-变形菌亚门(Uncultured Beta- proteobacterium)同源性分别达到99%和99%。G5与未培养碱菌属(Uncultured Alcaligenaceae bacterium)同源性达到97%。
Bacteria are both the producer and the decomposer in energy conversion and food chain of ecosystem; its diversity also has a variety of functions. Nitrogen is essential nutrients for plants, and is also an important limiting factor in soil productivity. The first step which is the rate-limiting step in nitrification, making NH4+ into NO2-, is completed by the ammonia-oxidizing bacteria (AOB).
Rhizosphere paddy soil is a special ecological environment and the microorganisms living there play an important role in the paddy production, soil structure and fertility maintenance, as well as the flow of nutrients. Study on these microorganisms and diversity of AOB may help us clarifying the interrelationship between soil environment, bacteria and AOB community structure; and it also could help us understanding the relationship among plants, soil and microorganisms. The research has significance in yields improving and environmental protection.
The fresh paddy soil in Wugong, Shaanxi province was sampling as test materials in this study. Total DNA of microorganism in rhizosphere paddy soil and non-rhizosphere paddy soil were extracted directly. Using universal primer pair 63F/1387R for bacteria and specific primers of ammonia-oxidizing bacteria Eub338/Nso1225, the partial gene fragments of 16S rDNA of bacteria and ammonia-oxidizing bacteria (AOB) were amplified by PCR, respectively, from the total DNA of rhizosphere paddy soil and non-rhizosphere paddy soil. The PCR fragments were connected with pMD19-T vector. After transforming these recombined plasmids into E coli JM109, the four clone libraries were set up. From each clone library about 150 inserted fragments of bacterial and AOB’s 16S rDNA were then amplified by PCR using universal primer M13 of pMD19-T vector and digested by restriction endonuclease Hha I and Rsa I, respectively.
The data got by PCR-RFLP was analyzed byα-measurement to figure out their bio-diversity characteristic; the clustering analysis was determined by software NTsys 2.10 with Bray-Curtis similarity as distance index which based on the distribution of bacteria and AOB. The preponderant clone of AOB in rhizosphere soil were selected for sequencing and BLASTN comparison on NCBI was used to get the high homology sequence and related information of strains. The phylogenetic trees of sequenced preponderant AOB were built by Cluster X and MEGA4.0.
Results showed that:
(1) Restriction endonuclease types of bacteria of rhizosphere paddy soil and non-rhizosphere soil were 124/141 and 116/140; the coverage (C value) of the clone libraries were 21.28% and 27.86%. Enzyme types of AOB in rhizosphere paddy soil and in non-rhizosphere soil were 110/167 and 105/155; the C value of the clone libraries were 51.70% and 52.26%.
(2) All the diversity data for bacteria was analyzed byα-measurement index, which showed that: Shannon-Wiener index (H′) were 4.764 and 4.658; Margalef richness index (d_(Ma)) were 24.855 and 23.272; Pielou evenness index (J_(gi)) were 0.988 and 0.980, for rhizosphere paddy soil and non-rhizosphere paddy soil. The results showed that in rhizosphere paddy soil the index value was larger than those in non-rhizosphere paddy soil; rhizosphere paddy soil has the bigger species abundance, the more equally distributing and microbial community with the better functional system.
(3) All the diversity data for AOB was analyzed byα-measurement index, which show that: Diversity index of rhizosphere paddy soil and non-rhizosphere paddy soil were got as 0.984 and 0.986 for Gini index (D_g), 0.952 and 0.966 for Evenness index (J_(gi)), 21.297 and 20.621 for Species richness index (d_(Ma)), and 4.701 and 4.654 for the largest species diversity index (H_(max)).
(4) By sequencing of the special bacteria G5, G13, G14, G26, G33, G42 and G111 in rhizosphere paddy soil, results showed that: G14 and G111 had a higher homology with Nitrosospira sp. which was up to 97% and 93%; G33 and G13 had a higher homology with Uncultured Nitrosomonadaceae bacterium which was up to 98% and 92%; G26 and G42 had a higher homology with Uncultured Beta proteobacterium which was up to 99% and 99%. G5 had a higher homology with Uncultured Alcaligenaceae bacterium which was up to 97%
水稻是人类赖以生存的粮食作物,水稻根际是一个特殊的生态环境,水稻根际微生物对于水稻的生产、土壤结构与肥力维持,以及在营养元素的流动过程中起着重要的作用。通过对水稻根际土细菌和氨氧化细菌多样性的研究,阐明根际土壤环境与细菌及氨氧化细菌群落结构之间的相互关系,可以加深人们对植物-土壤-微生物相互关系的理解和认识,对提高作物产量、保护环境等方面具有重要的意义。
本试验以陕西省武功县大田苗期水稻鲜土为研究对象,通过直接法提取根际水稻土和非根际水稻土微生物总DNA。采用细菌16S rDNA通用引物对(63F/1387R)和氨氧化细菌16S rDNA特异性引物(Eub338和Nso1225)分别对细菌及氨氧化细菌16S rDNA进行PCR扩增,将扩增片段与pMD19-T载体连接后转入大肠杆菌JM109中,通过蓝白斑筛选挑取阳性克隆子,分别建立水稻根际土和非根际土细菌、水稻根际土和非根际土氨氧化细菌等4个16S rDNA克隆文库。通过菌落PCR方法,从4个文库中分别随机挑取约150个阳性克隆,以pMD19-T载体通用引物M13重新扩增插入的16S rDNA片段,进行克隆文库的菌落PCR,将纯化后的菌落PCR产物分别用Hha I和Rsa I两种限制性内切酶水解,进行RFLP分型分析。
利用α多样性的测度分析RFLP的结果,聚类分析以细菌分布之间的Bray-Curtis相异性测度系数为距离指标,用非加权平均配对法(UPGA)在NTsys 2.10统计软件上进行数据处理。选取水稻根际土氨氧化细菌的优势克隆进行测序,所得的部分16S rDNA序列在NCBI上BLASTN比较,确定优势氨氧化细菌的种属;综合测序所得序列及NCBI上的相关序列,通过Cluster X及MEGA4.0构建进化树。
获得的主要试验结果如下:
(1)从水稻根际土中获得124个细菌的酶切类型,库容值为21.28%;从水稻非根际土中获得116个细菌的酶切类型,库容值为27.86%;从水稻根际土中获得110个氨氧化细菌的酶切类型,库容值为51.70%;从水稻非根际土中获得105个氨氧化细菌的酶切类型,库容值为52.26%。
(2)采用α多样性的测度对所得的细菌的RFLP分型数据进行分析,Shannon-Wiener(H′)多样性指数:水稻根际土为4.764,水稻非根际土为4.658;Margalef丰富度指数(d_(Ma)):水稻根际土为24.855,水稻非根际土为23.272;Pielou均匀度指数(J_(gi)):水稻根际土为0.988,水稻非根际土为0.980。其结果由大至小均为根际水稻土大于非根际水稻土。结果表明,水稻根际土中微生物物种丰富度较高,种类分布较均匀,构成功能体系较完善的微生物群落。
(3)采用α多样性的测度对所得的氨氧化细菌的RFLP分型数据进行分析,Gini(D_g)多样性指数:水稻根际土为0.984,水稻非根际土为0.986;均匀度指数(J_(gi)):水稻根际土为0.952,水稻非根际土为0.966;物种丰富度指数(d_(Ma)):水稻根际土为21.297,水稻非根际土为20.621;最大物种多样性指数(H_(max)):水稻根际土为4.701,水稻非根际土为4.654。
(4)根际水稻土优势氨氧化细菌G5、G13、G14、G26、G33、G42和G111的16S rDNA序列比对表明:G14和G111与亚硝化螺菌属(Nitrosospira sp.)同源性分别达到97%和93%; G33和G13与未培养亚硝化单胞菌属(Uncultured Nitrosomonadaceae bacterium)同源性分别达到98%和92%;G26和G42与未培养β-变形菌亚门(Uncultured Beta- proteobacterium)同源性分别达到99%和99%。G5与未培养碱菌属(Uncultured Alcaligenaceae bacterium)同源性达到97%。
Bacteria are both the producer and the decomposer in energy conversion and food chain of ecosystem; its diversity also has a variety of functions. Nitrogen is essential nutrients for plants, and is also an important limiting factor in soil productivity. The first step which is the rate-limiting step in nitrification, making NH4+ into NO2-, is completed by the ammonia-oxidizing bacteria (AOB).
Rhizosphere paddy soil is a special ecological environment and the microorganisms living there play an important role in the paddy production, soil structure and fertility maintenance, as well as the flow of nutrients. Study on these microorganisms and diversity of AOB may help us clarifying the interrelationship between soil environment, bacteria and AOB community structure; and it also could help us understanding the relationship among plants, soil and microorganisms. The research has significance in yields improving and environmental protection.
The fresh paddy soil in Wugong, Shaanxi province was sampling as test materials in this study. Total DNA of microorganism in rhizosphere paddy soil and non-rhizosphere paddy soil were extracted directly. Using universal primer pair 63F/1387R for bacteria and specific primers of ammonia-oxidizing bacteria Eub338/Nso1225, the partial gene fragments of 16S rDNA of bacteria and ammonia-oxidizing bacteria (AOB) were amplified by PCR, respectively, from the total DNA of rhizosphere paddy soil and non-rhizosphere paddy soil. The PCR fragments were connected with pMD19-T vector. After transforming these recombined plasmids into E coli JM109, the four clone libraries were set up. From each clone library about 150 inserted fragments of bacterial and AOB’s 16S rDNA were then amplified by PCR using universal primer M13 of pMD19-T vector and digested by restriction endonuclease Hha I and Rsa I, respectively.
The data got by PCR-RFLP was analyzed byα-measurement to figure out their bio-diversity characteristic; the clustering analysis was determined by software NTsys 2.10 with Bray-Curtis similarity as distance index which based on the distribution of bacteria and AOB. The preponderant clone of AOB in rhizosphere soil were selected for sequencing and BLASTN comparison on NCBI was used to get the high homology sequence and related information of strains. The phylogenetic trees of sequenced preponderant AOB were built by Cluster X and MEGA4.0.
Results showed that:
(1) Restriction endonuclease types of bacteria of rhizosphere paddy soil and non-rhizosphere soil were 124/141 and 116/140; the coverage (C value) of the clone libraries were 21.28% and 27.86%. Enzyme types of AOB in rhizosphere paddy soil and in non-rhizosphere soil were 110/167 and 105/155; the C value of the clone libraries were 51.70% and 52.26%.
(2) All the diversity data for bacteria was analyzed byα-measurement index, which showed that: Shannon-Wiener index (H′) were 4.764 and 4.658; Margalef richness index (d_(Ma)) were 24.855 and 23.272; Pielou evenness index (J_(gi)) were 0.988 and 0.980, for rhizosphere paddy soil and non-rhizosphere paddy soil. The results showed that in rhizosphere paddy soil the index value was larger than those in non-rhizosphere paddy soil; rhizosphere paddy soil has the bigger species abundance, the more equally distributing and microbial community with the better functional system.
(3) All the diversity data for AOB was analyzed byα-measurement index, which show that: Diversity index of rhizosphere paddy soil and non-rhizosphere paddy soil were got as 0.984 and 0.986 for Gini index (D_g), 0.952 and 0.966 for Evenness index (J_(gi)), 21.297 and 20.621 for Species richness index (d_(Ma)), and 4.701 and 4.654 for the largest species diversity index (H_(max)).
(4) By sequencing of the special bacteria G5, G13, G14, G26, G33, G42 and G111 in rhizosphere paddy soil, results showed that: G14 and G111 had a higher homology with Nitrosospira sp. which was up to 97% and 93%; G33 and G13 had a higher homology with Uncultured Nitrosomonadaceae bacterium which was up to 98% and 92%; G26 and G42 had a higher homology with Uncultured Beta proteobacterium which was up to 99% and 99%. G5 had a higher homology with Uncultured Alcaligenaceae bacterium which was up to 97%
引文
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