加拿大草原地区不同土壤类型的丛枝菌根真菌（AMF）遗传多样性研究
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摘要
丛枝菌根真菌(arbuscular mycorrhizal fungi, AMF)是土壤中广泛分布并能与绝大部分维管束植物根系形成互利共生的关系,因此成为自然生态系统中重要的组成部分。AMF对宿主植物优良的共生效益是人们研究它的主要兴趣所在,希望掌握AMF对宿主植物的促生效应达到合理和有效利用菌根资源的目的。然而AMF是一种活体共生真菌,只有在生活的植物根系中才能完成生活史,目前AMF纯培养一直是未能解决,致使人们对它的生理生化代谢,遗传学,尤其是其自然资源多样性的了解十分有限。目前新发展的第二代测序技术(the next generation sequencing)如焦磷酸测序技术,较传统的克隆和测序方法具有更加高效、准确和高信息量的优势。但巢式PCR由于其较多的扩增循环数,并不适合以焦磷酸测序为基础的方法。因为相对增多的假阳性序列比例会产生更多的假阳性测序结果,将强烈影响到最后对AMF物种丰富度和多样性的测算和估计。
本论文通过测试不同的目的基因扩增方法,优化PCR扩增体系和目的产物纯化体系,探索出适合从土壤总DNA中扩增AMF目的基因并用于焦磷酸测序的方案。现对各部分实验进行如下总结和概括：
1.不同DNA样品处理方法对焦磷酸测序结果的影响。首先设计该实验方案,分别比较巢式PCR和单一PCR扩增DNA样品后对焦磷酸测序结果的不同影响,并利用PCR产物纯化试剂盒代替传统的凝胶电泳分离产物,切胶提纯的方法,以期找到适合焦磷酸测序的扩增DNA样品的最优方案。该实验10个土壤样品分别来自不同小麦大田,首先从土壤中提取总DNA,再用AMF特异引物扩增AMF目的基因——核糖体小亚基(18S rDNA),该基因结构简图如下：核糖体重复基因序列一段核糖体基因重(?)复序列放大图SSU (18S)5.8S ITS LSU (25-28S)5S RNA RNA RNA RNA
图1核糖体目的基因简图。(仿Vilgalys实验室)
我们设计了三种方法来扩增原始DNA和纯化目的产物。方法一,巢式PCR扩增目的基因(先用真菌通用引物扩增土壤中的所有真菌18S rDNA基因,再用AMF特异引物扩增AMF的18S rDNA基因),凝胶电泳分离并纯化目的DNA片段。方法二,单一PCR并重复三次扩增目的基因,凝胶电泳分离并纯化目的DNA片段。方法三,单一PCR并重复三次扩增目的基因,PCR纯化试剂盒通过电荷交换原理直接纯化目的DNA片段。三种方法处理的10个DNA样品同时送样进行焦磷酸测序,测序结果显示：在97%相似度水平上,方法一共获得1319条有效序列,其中209条是AMF序列,产生25个AMF分子分类单位(OTU)；方法二共获得7222条有效序列,其中931条属于AMF,产生89个OTU；方法三获得80875条有效序列,14736属于AMF,产生410个OTU,且方法一和方法二中大多数产生的OTU都包括在方法三中。通过该研究探索,我们不仅首次成功地证实了单一PCR适宜于焦磷酸测序方法,而且通过对比证实了PCR产物纯化试剂盒比传统切胶纯化方法更加有效回收目的DNA片段,还支持了重复扩增同一样品对有效获得目的基因片段的观点,即对同一DNA样品进行3-5次重复扩增,再混合所有的重复扩增产物,增加目的基因的得率。
2.焦磷酸测序法调查AMF多样性及其系统学研究。在AMF自然资源的调查方面,目前对于这方面的了解很有局限性,这也是阻碍对AMF有效利用的主要原因。研究背景是北美中部农业产地,主要集中在加拿大草原三省,这里农作物生产是其主要的的支柱产业,如能对菌根进行有效利用无疑是对农作物产量和品质有力保障。我们对76个小麦大田采样点的AMF生物群落结构进行了调查,总采样面积覆盖了2800多万公顷加拿大草原地区,采用DNA条形码标记的高通量焦磷酸测序方法。结果共测得33个AMF分子分类单位(OTU),系统学分析显示14个OTU归于Funneliformis和Rhizophagus属,16个OTU归于Claroideoglomus属,还有3个是多孢囊霉属(Diversisporales)。多重反应行排序(multiresponse permutation procedure, MRPP)分析显示虽然有些AMF种类在各个土壤类型中均有分布,但其分布特点受土壤类型的影响(P=0.04),其中黑钙土(Black Chernozem)中含有最丰富和多样的AMF种类。
3.AMF生态分布特点及土壤环境影响因素研究。大量研究表明AMF资源分布受环境因素的影响,如土壤pH值可以影响菌根侵染率和种类多样性。本论文的第三部分研究结合实际测量的土壤营养元素指标和土壤物理性质,找出影响菌根真菌分布的具体土壤环境因素。主要检测内容有：四种耕作土壤类型——黑钙土(Black Chernozem)、褐钙土(Brown Chernozem)、深褐钙土(Dark Brown Chernozem)、灰钙土(Gray Chernozem)的基本生化性质检测；利用植物根系与土壤离子交换生化信号传感器(Plant Root Sensor, PRS)检测土壤主要营养元素N、P、K,微量元素测定(Ca、Mg、Fe、Mn、Cu、 Zn、B、S、Al)以及有害物质Pb、Cd的检测；土壤pH值、土壤颗粒密度、速效磷、总氮含量的测定；土壤菌丝量以及小麦根侵染率测定；小麦地上部分生物量和组织N、P含量的测定；对AMF目的基因(18S rDNA)扩增及焦磷酸测序。数理统计通过冗余分析发现：AMF资源分布与不同土壤类型显著相关(P=0.036),在黑土和深褐土中菌根资源多样性和丰富度较高,褐土最低；AMF种群与土壤营养元素含量显著相关(P=0.005); AMF能显著促进植物营养元素的吸收(P=0.047),特别是Cu, Zn等微量元素的吸收；AMF侵染率与土壤磷含量呈显著负相关(P=0.0002)。
Arbuscular mycorrhizal fungi (AMF) are kind of the popular mycorrhizal fungi, which could colonize most of the terrestrial vascular plant on the earth, and play an important role in the natural ecosystem, that is the reason of people interesting in AMF research. In order to rationally take advantage of AMF, we need to understand how these fungi could improve growth of plant. However, AMF can not grow in vitro, which limited their study of metabolizable physiology, biochemistry and genetic knowledge, especially on the aspect of their diveristy of natural resource. Recently developed several effective and short time consume methods with high-throughput base sequences and high accuracy produced which represented by the next generation sequencing (named454pyrosequencing hereafter). While nested-PCR works well for amplifying little amount of mycorrhizal fungi from environmental samples but might not good procedure for downstream pyrosequencing owing to accumulated PCR cycles is one of the probable causes of sequence errors and chimeras which strongly impact the estimates of diversity and richness.
This study tested different methods for amplifying target gene, and purifying PCR amplicons, thus modify the optimum protocol of yield AMF target DNA fragment for pyrosequencing. Every experiments of this study were stated below in details:
1. Effect of different ways to process raw DNA in soil for pyrosequencing. Here, we made test to compare the difference of using nest-PCR and PCR protocol to amplify mycorrhizal fungi DNA from the same soil samples respectively, and effect of PCR-induced sequence bias and assessing fungal community structure and richness.10commercial wheat fields were selected for soil sampling and18S rDNA was considered as target DNA region (Fig.1).
Fig.1Map of ribosome repeat unit gene.(Source:Vilgalys lab)
We designed three different DNA processing methods:Method Ⅰ using nested-PCR within agarose gel for purifying PCR products, Method Ⅱ using simple PCR with multiple reactions also within agarose gel for purification and Method Ⅲ adopt simple PCR with parallel replicated reactions follow by magnetic beads using charge switch principle for PCR purification. Results show that at97%similarity level Method Ⅲ detected valid sequences (80875),14736of which are mycorrhizal fungal sequences and generated410AMF operational taxonomic units (OTUs); the other two methods (for method Ⅰ, there are totally1319sequences and209of which belong to AM fungi with25AM fungal OTUs. For Method Ⅱ, totally7222sequences and931of which were identified AM fungi with89OTUs) were much less than Method Ⅲ. Almost OTUs detected by Method Ⅰ and Ⅱ were shared by Method Ⅲ. In order to explore the optimal PCR amplicons for pyrosequencing with time saving method, we also using PCR purification kit instead of running agarose gel electrophoresis to clear PCR products as another part of test work. We hypothesized that ⅰ) run parallel PCR replica and then pool into large volume for subsequent concentration will obtain sufficient mycorrhizal fungal DNA yield. ⅱ) nest-PCR will generate more based-sequence artifacts than simply PCR protocol. ⅲ) PCR purification kit is a good choice of processing PCR amplicon for enough DNA quantities and time saving.
2. Study of AMF diveristy and phylogenetic analysis by pyrosequencing. Insufficient knowledge of the AM fungal resources naturally present in the landscape is a major impediment to the management of AM symbioses in crop production in the Canadian Prairie provinces where an important agricultural industry has developed over the last century. The composition of the AM fungal communities in76wheat fields distributed over28million ha in the Canadian Prairie was described using a tag-encoded massively parallel pyrosequencing protocol. Of the33dominant AMF operational taxonomic units (AMF OTUS) found in the76wheat fields surveyed at anthesis in2009,14clustered as Funneliformis/Rhizophagus,16as Claroideoglomus, and3as Diversisporales. Multiresponse permutation procedure (MRPP) analysis showed that AMF OTUS community composition was better explained by the Chernozem great groups (P=0.0044) than by measured soil properties. Black Chernozem is the most conducive to AMF proliferation. AMF are generally distributed according to Chernozem great groups in the Canadian Prairie, although some taxa are evenly distributed in all soil groups.
3. Study of AMF distribution and associated environmental factors. Lots of research indicated that environmental factors signifcantly effected AMF distribution, such as soil pH can affect AM functional diveristy by inducing changes in root colonization and species diveristy. The third part of this study is on the perspective that distribtuion and richness of AMF community could be affected by soil nutrients and properties. The content of research work included soil description for the four soil types (Black Chernozem, Brown Chernozem, Dark Brown Chernozem, Gray Chernozem), soil nutrients (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B, S, Al, Pb, Cd), soil pH, bulk density, Olsen P, hyphal trap, AM root colonization, wheat dry biomass, wheat tissue N, P and pyrosequencing for AMF18S rDNA. Redundancy analysis (RDA) found that AMF distribution was significantly affect by soil type (P=0.036), Black and Dark Brown Chernozem have higher levels of AMF diversity and richness,while Brown Chernozem was the lowest. AMF community was strongly affected by soil nutrients (P=0.005). AMF contributed plant nutrients uptake (e.g., Cu, Zn)(P=0.047). This study also found that soil P was negatively correlated with AMF root colonization (P=0.0002).
本论文通过测试不同的目的基因扩增方法,优化PCR扩增体系和目的产物纯化体系,探索出适合从土壤总DNA中扩增AMF目的基因并用于焦磷酸测序的方案。现对各部分实验进行如下总结和概括：
1.不同DNA样品处理方法对焦磷酸测序结果的影响。首先设计该实验方案,分别比较巢式PCR和单一PCR扩增DNA样品后对焦磷酸测序结果的不同影响,并利用PCR产物纯化试剂盒代替传统的凝胶电泳分离产物,切胶提纯的方法,以期找到适合焦磷酸测序的扩增DNA样品的最优方案。该实验10个土壤样品分别来自不同小麦大田,首先从土壤中提取总DNA,再用AMF特异引物扩增AMF目的基因——核糖体小亚基(18S rDNA),该基因结构简图如下：核糖体重复基因序列一段核糖体基因重(?)复序列放大图SSU (18S)5.8S ITS LSU (25-28S)5S RNA RNA RNA RNA
图1核糖体目的基因简图。(仿Vilgalys实验室)
我们设计了三种方法来扩增原始DNA和纯化目的产物。方法一,巢式PCR扩增目的基因(先用真菌通用引物扩增土壤中的所有真菌18S rDNA基因,再用AMF特异引物扩增AMF的18S rDNA基因),凝胶电泳分离并纯化目的DNA片段。方法二,单一PCR并重复三次扩增目的基因,凝胶电泳分离并纯化目的DNA片段。方法三,单一PCR并重复三次扩增目的基因,PCR纯化试剂盒通过电荷交换原理直接纯化目的DNA片段。三种方法处理的10个DNA样品同时送样进行焦磷酸测序,测序结果显示：在97%相似度水平上,方法一共获得1319条有效序列,其中209条是AMF序列,产生25个AMF分子分类单位(OTU)；方法二共获得7222条有效序列,其中931条属于AMF,产生89个OTU；方法三获得80875条有效序列,14736属于AMF,产生410个OTU,且方法一和方法二中大多数产生的OTU都包括在方法三中。通过该研究探索,我们不仅首次成功地证实了单一PCR适宜于焦磷酸测序方法,而且通过对比证实了PCR产物纯化试剂盒比传统切胶纯化方法更加有效回收目的DNA片段,还支持了重复扩增同一样品对有效获得目的基因片段的观点,即对同一DNA样品进行3-5次重复扩增,再混合所有的重复扩增产物,增加目的基因的得率。
2.焦磷酸测序法调查AMF多样性及其系统学研究。在AMF自然资源的调查方面,目前对于这方面的了解很有局限性,这也是阻碍对AMF有效利用的主要原因。研究背景是北美中部农业产地,主要集中在加拿大草原三省,这里农作物生产是其主要的的支柱产业,如能对菌根进行有效利用无疑是对农作物产量和品质有力保障。我们对76个小麦大田采样点的AMF生物群落结构进行了调查,总采样面积覆盖了2800多万公顷加拿大草原地区,采用DNA条形码标记的高通量焦磷酸测序方法。结果共测得33个AMF分子分类单位(OTU),系统学分析显示14个OTU归于Funneliformis和Rhizophagus属,16个OTU归于Claroideoglomus属,还有3个是多孢囊霉属(Diversisporales)。多重反应行排序(multiresponse permutation procedure, MRPP)分析显示虽然有些AMF种类在各个土壤类型中均有分布,但其分布特点受土壤类型的影响(P=0.04),其中黑钙土(Black Chernozem)中含有最丰富和多样的AMF种类。
3.AMF生态分布特点及土壤环境影响因素研究。大量研究表明AMF资源分布受环境因素的影响,如土壤pH值可以影响菌根侵染率和种类多样性。本论文的第三部分研究结合实际测量的土壤营养元素指标和土壤物理性质,找出影响菌根真菌分布的具体土壤环境因素。主要检测内容有：四种耕作土壤类型——黑钙土(Black Chernozem)、褐钙土(Brown Chernozem)、深褐钙土(Dark Brown Chernozem)、灰钙土(Gray Chernozem)的基本生化性质检测；利用植物根系与土壤离子交换生化信号传感器(Plant Root Sensor, PRS)检测土壤主要营养元素N、P、K,微量元素测定(Ca、Mg、Fe、Mn、Cu、 Zn、B、S、Al)以及有害物质Pb、Cd的检测；土壤pH值、土壤颗粒密度、速效磷、总氮含量的测定；土壤菌丝量以及小麦根侵染率测定；小麦地上部分生物量和组织N、P含量的测定；对AMF目的基因(18S rDNA)扩增及焦磷酸测序。数理统计通过冗余分析发现：AMF资源分布与不同土壤类型显著相关(P=0.036),在黑土和深褐土中菌根资源多样性和丰富度较高,褐土最低；AMF种群与土壤营养元素含量显著相关(P=0.005); AMF能显著促进植物营养元素的吸收(P=0.047),特别是Cu, Zn等微量元素的吸收；AMF侵染率与土壤磷含量呈显著负相关(P=0.0002)。
Arbuscular mycorrhizal fungi (AMF) are kind of the popular mycorrhizal fungi, which could colonize most of the terrestrial vascular plant on the earth, and play an important role in the natural ecosystem, that is the reason of people interesting in AMF research. In order to rationally take advantage of AMF, we need to understand how these fungi could improve growth of plant. However, AMF can not grow in vitro, which limited their study of metabolizable physiology, biochemistry and genetic knowledge, especially on the aspect of their diveristy of natural resource. Recently developed several effective and short time consume methods with high-throughput base sequences and high accuracy produced which represented by the next generation sequencing (named454pyrosequencing hereafter). While nested-PCR works well for amplifying little amount of mycorrhizal fungi from environmental samples but might not good procedure for downstream pyrosequencing owing to accumulated PCR cycles is one of the probable causes of sequence errors and chimeras which strongly impact the estimates of diversity and richness.
This study tested different methods for amplifying target gene, and purifying PCR amplicons, thus modify the optimum protocol of yield AMF target DNA fragment for pyrosequencing. Every experiments of this study were stated below in details:
1. Effect of different ways to process raw DNA in soil for pyrosequencing. Here, we made test to compare the difference of using nest-PCR and PCR protocol to amplify mycorrhizal fungi DNA from the same soil samples respectively, and effect of PCR-induced sequence bias and assessing fungal community structure and richness.10commercial wheat fields were selected for soil sampling and18S rDNA was considered as target DNA region (Fig.1).
Fig.1Map of ribosome repeat unit gene.(Source:Vilgalys lab)
We designed three different DNA processing methods:Method Ⅰ using nested-PCR within agarose gel for purifying PCR products, Method Ⅱ using simple PCR with multiple reactions also within agarose gel for purification and Method Ⅲ adopt simple PCR with parallel replicated reactions follow by magnetic beads using charge switch principle for PCR purification. Results show that at97%similarity level Method Ⅲ detected valid sequences (80875),14736of which are mycorrhizal fungal sequences and generated410AMF operational taxonomic units (OTUs); the other two methods (for method Ⅰ, there are totally1319sequences and209of which belong to AM fungi with25AM fungal OTUs. For Method Ⅱ, totally7222sequences and931of which were identified AM fungi with89OTUs) were much less than Method Ⅲ. Almost OTUs detected by Method Ⅰ and Ⅱ were shared by Method Ⅲ. In order to explore the optimal PCR amplicons for pyrosequencing with time saving method, we also using PCR purification kit instead of running agarose gel electrophoresis to clear PCR products as another part of test work. We hypothesized that ⅰ) run parallel PCR replica and then pool into large volume for subsequent concentration will obtain sufficient mycorrhizal fungal DNA yield. ⅱ) nest-PCR will generate more based-sequence artifacts than simply PCR protocol. ⅲ) PCR purification kit is a good choice of processing PCR amplicon for enough DNA quantities and time saving.
2. Study of AMF diveristy and phylogenetic analysis by pyrosequencing. Insufficient knowledge of the AM fungal resources naturally present in the landscape is a major impediment to the management of AM symbioses in crop production in the Canadian Prairie provinces where an important agricultural industry has developed over the last century. The composition of the AM fungal communities in76wheat fields distributed over28million ha in the Canadian Prairie was described using a tag-encoded massively parallel pyrosequencing protocol. Of the33dominant AMF operational taxonomic units (AMF OTUS) found in the76wheat fields surveyed at anthesis in2009,14clustered as Funneliformis/Rhizophagus,16as Claroideoglomus, and3as Diversisporales. Multiresponse permutation procedure (MRPP) analysis showed that AMF OTUS community composition was better explained by the Chernozem great groups (P=0.0044) than by measured soil properties. Black Chernozem is the most conducive to AMF proliferation. AMF are generally distributed according to Chernozem great groups in the Canadian Prairie, although some taxa are evenly distributed in all soil groups.
3. Study of AMF distribution and associated environmental factors. Lots of research indicated that environmental factors signifcantly effected AMF distribution, such as soil pH can affect AM functional diveristy by inducing changes in root colonization and species diveristy. The third part of this study is on the perspective that distribtuion and richness of AMF community could be affected by soil nutrients and properties. The content of research work included soil description for the four soil types (Black Chernozem, Brown Chernozem, Dark Brown Chernozem, Gray Chernozem), soil nutrients (N, P, K, Ca, Mg, Fe, Mn, Cu, Zn, B, S, Al, Pb, Cd), soil pH, bulk density, Olsen P, hyphal trap, AM root colonization, wheat dry biomass, wheat tissue N, P and pyrosequencing for AMF18S rDNA. Redundancy analysis (RDA) found that AMF distribution was significantly affect by soil type (P=0.036), Black and Dark Brown Chernozem have higher levels of AMF diversity and richness,while Brown Chernozem was the lowest. AMF community was strongly affected by soil nutrients (P=0.005). AMF contributed plant nutrients uptake (e.g., Cu, Zn)(P=0.047). This study also found that soil P was negatively correlated with AMF root colonization (P=0.0002).
引文
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