应用克隆文库构建法研究枇杷根系AMF多样性  

Study on Species Diversity of AMF Community Colonizing Loquat Roots by Constructing Clone Library

作　　者：尹利方 任禛[1,2,3] 夏体渊[1] 朱维贤[4] 陈泽斌[1,2] 靳松[1] 颜家亮 赵晓红[1] 

机构地区：[1]昆明学院农学院,云南昆明650214 [2]云南省都市特色农业工程技术研究中心,云南昆明650214 [3]云南省高校特色生物资源开发与利用重点实验室,云南昆明650214 [4]昆明市农业科学研究院,云南昆明650034

出　　处：《西南农业学报》2017年第5期1009-1015,共7页Southwest China Journal of Agricultural Sciences

基　　金：云南省都市特色农业工程技术研究中心项目(TSNY0201);云南省教育厅科学研究基金项目(2014Y390);云南省应用基础研究青年项目(2013FD040);云南省应用基础研究自筹项目(2013FZ099);昆明学院人才引进项目(YJL14005);昆明学院人才引进基金项目(YJL12010);昆明学院校立重点基金项目(XJL12020);昆明学院大学生科研项目(XJD16081);云南中烟品牌省内原料产地生态与主栽品种合理布局研究项目;云南省高校优势特色重点学科(生态学)建设项目资助(05000511311);昆明市农业局滇池流域农业面源污染综合防控对策研究(2016JC01);云南省高校特色生物资源开发与利用重点实验室开放基金项目(GXKJ201623)

摘　　要：【目的】为进一步探寻和利用宝贵的AMF资源提供有效的研究方法。【方法】在昆明学院随机采集枇杷根系作为实验样品,采用CTAB法提取根系DNA,进行18S r DNA PCR扩增,产物连接转入大肠杆菌JM109受体,建立18S rRNA部分基因克隆文库。从文库中随机选取35个白色克隆子,经鉴定,获得30个阳性克隆子,将阳性克隆子测序。运用Mothur等软件分析阳性克隆子序列,确定9条差异序列,为不同的AMF序列。【结果】克隆文库的Coverage C值为93.3%,克隆文库的Shannon-Wiener指数为2.079,Simpson指数为0.87,且Rarefaction曲线比较饱和。9个序列分为了两个不同的类群,代表不同AMF种类;球囊霉属(Glomus)是枇杷根系的主要AMF类群,OUT1和OTU2代表了文库中的主要类型,其中OTU1为根内根生囊霉(Rhizophagus irregularis);OTU3、OTU4、OTU5和OTU6代表着文库中的常见类型;其余序列则为枇杷根系中的少见或稀有AMF类型。【结论】球囊霉属(Glomus)是枇杷根系的主要AMF类型,其中根内根生囊霉(Rhizophagus irregularis)为优势AMF种类。[ Objective] This study aimed to find the effective research methods for exploring and utilizing valuable AMF resources. [ Method] The roots of loquat were collected randomly as experimental samples from Kunming University. The 18S rRNA partial gene clone library was established by connected DNA which used the CTAB method and amplified to Escherichia coil JM109. 30 positive clones that were selected randomly from 35 white clones in the library were sequenced. The Mothur software analyzed the sequence of positive clones, and got 9 differ-ent AMF sequences. [ Result ] The Coverage C of the clone library was 93.3 %. The Shannon-Wiener was 2. 079, and the Simpson index was O. 87 in the clone library, the curve was sat- urated. The 9 sequences were divided into two distinct groups representing different AMF species; Glomus was the main AMF groups of loquat root. OUT1 and OTU2 represented the main types of the library, here OTU1 was Rhizophagus irregularis. OTU3, OTU4, OTU5 and OTU6 represented the common types of the library; the rest sequences were rare AMF types. [ Conclusion ] Glomus is the main type of AMF in the roots of loquat, in which Rhizophagus irregularis is the dominant AMF species.

关 键 词：枇杷 AMF 克隆文库 18S RRNA基因 系统发育分析 

分 类 号：S667.3[农业科学—果树学]