《（生物科技行业）喜树替代紫茎泽兰过程中根际微生物群落特征》由会员分享，可在线阅读，更多相关《（生物科技行业）喜树替代紫茎泽兰过程中根际微生物群落特征（11页珍藏版）》请在金锄头文库上搜索。
1、喜树替代紫茎泽兰过程中根际微生物群落特征收稿日期: 2006* 基金项目:国家林业局林业有害植物调查专项资助，教育部重点项目（104191）,资助* 联系人: Tel: 0451-82191517祖元刚1, 高崇洋1, 王文杰1, 杨逢建1，刘英1, 王敏1 , 赵阳国2(1东北林业大学森林植物生态学教育部重点实验室，哈尔滨 150040; 2 哈尔滨工业大学市政环境工程学院，哈尔滨 150090)摘要：为研究我国重要经济树种喜树根际分泌物对根际微生物群落的影响，以及喜树在控制林业有害植物紫茎泽兰生物入侵的可行性，采用传统培养技术和分子生物学技术PCR-单链构象多态性(SSCP)、末端限制性片
2、段长度多态性(TRFLP)-16S rDNA文库相结合对喜树(Ca)、紫茎泽兰(Ea)和二者混栽体系(CE)根际土壤中的真核微生物和真细菌微生物群落结构进行了比较。计数结果表明喜树、二者混栽以及紫茎泽兰根际中的真细菌数量依次减少而真核微生物数量依次增多。PCR-SSCP分析显示，紫茎泽兰根际土壤中真核微生物条带数量(多样性)远高于喜树和混栽根际，对部分条带克隆测序表明喜树根际中Meristolohmannia spp.为主要优势种群；TRFLP对真细菌结构分析表明，三种根际中真细菌群落多样性丰富，但没有差别，对喜树根际真细菌16S rDNA文库测序比较分析，共包含了10个已分类的门，其中Pro
3、teobacteria 门为优势菌群，占24.71%(其中-Proteobacteria 占17.65%)，Acidobacteria 门占16.47%，Bacteroidetes 门占10.59%。另外，色谱分析发现喜树和混栽根际土壤中分别含有较低浓度喜树碱和羟基喜树碱，而紫茎泽兰根际二者均检测不到。由此可见，紫茎泽兰的扩散和蔓延依赖于特有的真核微生物群落结构模式，并不改变真细菌群落的结构，喜树能够通过根系分泌物改变紫茎泽兰根际真核微生物群落结构模式，进而制约其外延。本研究将为喜树在紫茎泽兰生物替代中提供理论基础。关键词：喜树，根际微生物，紫茎泽兰，PCR-单链构象多态性技术(SSCP)，末
4、端限制性片段长度多态性(TRFLP)，16S rDNA文库Characteristics of Microbial Community in Rhizosphere of Camptotheca Acuminata Mix-Cultured with Exotic Invasive Plant Eupatorium adenophorumZU Yuan-gang1, GAO Chong-yang1, WANG Wen-jie1, Yang Fengjian1, Liu Ying1, Wang Min1, ZHAO Yang-guo2(1Key Laboratory of Forest Plan
5、t Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040; 2 School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090)Abstract: In order to investigate the impacts of secretion from Camptotheca acuminate (Ca) roots on the quantities and st
6、ructure of Fungi and Bacteria in the rhizosphere, and the possibility that Ca controls exotic invasive plant Eupatorium adenophorum (Ea), traditional culture-dependent counting method and culture-independent small-subunit-ribosomal RNA gene-targeted molecular techniques, PCR-Single-Strand Conformati
7、on Polymorphism (SSCP), terminal Restraion Fragement Length Polymorphism(TRFLP) combined with 16S rDNA library, were adopted to compare the composition and structure of Fungi and Bacteria in rhizospheres of Ca, Ea and Ca-Ea mixed plantation(CE). Counting results indicated the numbers of Bacteria inc
8、reased in turns in rhizospheres of Ea, Ca-Ea and Ca, while that of Fungi decreased in turns. PCR-SSCP profiles showed eucaryotic microbial bands (corresponding to biodiveristy) in rhizosphere of Ea were more complex than that of Ca and CE. It was Meristolohmannia spp. that became the sole predominan
9、t group in the rhizosphere of Ca demonstrated by cloning and sequencing a part of main SSCP bands. TRFLP analysis on Bacteria showed no difference among 3 rhizospheres, and the sequences of 16S rDNA clone library from Ca rhizospheres distributed in 10 known phyla, in which phylum Proteobacteria was
10、the absolute dominant group and accounted for 24.71% of cloned sequences (-Proteobacteria accounted for up to 17.65%), and phyla Acidobacteria and Bacteroidetes accounted for 16.47% and 10.59% of cloned sequences respectively. In addition, high performance liquid chromatography checked out a trace a
11、mount of camptothecin and hydroxycamptothecin in the rhizospheric soil of Ca and CE, but examined no either camptothecin or hydroxycamptothecin in rhizospheric soil of Ea. Therefore, invasion and diffusion of Ea evidently depended on distinctive eukaryotic community structure mode, but not on the ba
12、cterial mode. And Ca was able to alter the invasive eukaryotic community structure mode of Ea by secreting camptothecin and hydroxycamptothecin into rhizospheres, and further inhitibited overspread of Ea. This study provided rhizospheric theoretic evidence for substituting Ca for Ea.Key words: Campt
13、otheca acuminate, rhizospheric microbe, Eupatorium adenophorum, PCR-Single-Strand Conformation Polymorphism (SSCP), terminal Restraion Fragement Length Polymorphism (TRFLP), 16S rDNA library 喜树(Camptotheca acuminata Decaisne, Ca)是多年生亚热带落叶阔叶树，因其能够合成显著抗肿瘤活性的次生代谢产物喜树碱(camptothecin)和羟基喜树碱(hydroxycamptot
14、hecin)而倍受人们的关注。文献表明，喜树碱能够与人的拓扑异构酶I-DNA (TopI-DNA )共价复合物结合，抑制酶在超螺旋双链DNA上形成缺刻，从而阻止DNA的解旋，进而起到抑制肿瘤细胞复制的抗癌作用1。喜树碱在嫩叶中的含量为种子中的1.5倍，树皮中的2.5倍2,有研究表明喜树根际也能够分泌喜树碱3，由于喜树碱几乎对所有真核生物均具有抑制作用，所以喜树可能对根际土壤中的真核微生物群落结构产生很大的影响。紫茎泽兰(Eupatorium adenophorum, Ea)作为重要的外来入侵植物越来越受到人们的重视，而对入侵植物的生物防治是最具应用潜力的策略之一。研究表明，紫茎泽兰等许多外来植
15、物的入侵是通过改变根际中微生物群落的功能来实现的4-8，通过改变土壤中原有微生物群落模式来达到成功扩散的目的。而喜树根际能够分泌抑制真核微生物生长的物质从而极有可能改变整个微生物群落的结构，进而破坏紫茎泽兰根际周围极具扩散性的微生物群落模式，从而实现对这种入侵植物的成功替代。根际中的微生物由于与宿主植物之间的相互依赖，相互制约的关系，致使不同植物根际的微生物群落具有很高的特异性9,10，喜树作为重要经济树种，其根际微生物的群落结构也应存在其特异之处，而以培养技术进行分析是无法满足对精确，客观，快速的要求。末端限制性片段长度多态性(terminal Restraion Fragement Len
16、gth Polymorphism,TRFLP)11和PCR-单链构象多态性(PCR-Single-Strand Conformation Polymorphism PCR-SSCP) 12技术作为重要的基因指纹技术，在揭示微生物群落结构与动态特征中应用越来越广泛。而真细菌16S rDNA文库技术能更全面的揭示了真细菌微生物多样性，Giovannoni等13首先采用该技术分析马尾藻海海面上浮游微生物的多样性，发现了一个新的且在该环境中占优势的微生物类群。目前，该技术已经广泛运用于土壤、海洋、湖泊、动物肠道等多种生态系统中微生物多样性的调查，揭示了环境当中前所未知的微生物的多样性。本研究采用传统培养技术对紫茎泽兰、紫茎泽兰-喜树、喜树三种根际中微生物数量进行了调查，并采用SSCP和TRFLP-16S rDNA文库技术对三种根际中的真核微生物和真细菌的多样性、相似性进行了比较，对喜树控制紫茎泽兰入侵和扩散的可行性进行了分析。1 材料与方法1.1 实验设计选择
