【作者】 赵红敬；

【导师】 高必达； 朱水芳； 徐涛；

【作者基本信息】 湖南农业大学 ， 生物安全与检疫， 2011， 硕士

【摘要】 水稻是禾本科中很重要的粮食作物,养活着世界上大部分人口。水稻中含有丰富的种质资源,而目前由于人为破坏活动及缺乏相应的检验技术,致使我国水稻资源流失严重,造成巨大的损失。对稻种资源及多样性及时进行有效的保护,是保证我国水稻品质改善和持久生产的基础。为建立快速、高效的重要水稻资源的分子查验技术体系,本研究共收集了普通野生稻,药用、斑点、疣粒、高杆、宽叶、紧穗和长雄蕊8种类型的野生稻以及2种类型的栽培稻材料共计105份,采用基因条码技术及dCAPS分子标记旨在建立对野生稻、栽培稻等种的特异性进行有效的区分的资源查验技术体系。1.采用基因条码技术,对水稻材料的matK、trnL、rpoB、rpoC、Nadl、ndhJ、atpF-atpH、psbK-psb1、Adh2、Os9971进行扩增并自行测序,对测序结果较好的trnL、rpoB、rpoC、Nad1、ndhJ、atpF-atpH、psbK-psbl7个条码基因,结合genebank已公布的基因序列信息,利用MEGA4软件进行系统进化亲缘关系分析,得出以下结论：(1)trnL基因具有较多的多态性位点,不仅能将禾本科作物中属级作物区分开,而且能定位至基因组型,将所分析的36份水稻材料共分为AA,CC, DD,CCDD四个染色体组型(2)通过对Nadl与psbK-psbl两个基因的分析,表明了宽叶和高杆野生稻与其他水稻材料相比具有较近的亲缘关系。(3)atpF-atpH、rpoB、rpoC、ndhJ等条码基因鉴于非常保守的特点,比较适合做属以上的分类结元进行鉴定。2.探讨了基于SNP位点建立dCAPS分子标记体系。采用生物信息学软件寻找了药用野生稻和斑点野生稻上trnL、rpoB、rpoC、matK、atpF-atpH、psbK-psb1 ndhJ8个基因碱基序列上适合设计错配引物的SNP位点,设计合成适当的错配引物进行PCR扩增,选择相应的限制性内切酶进行酶切。在这2种野生稻DNA的matK基因上分别找到1个合适的SNP位点,设计了3对错配引物,斑点野生稻的trnL基因上找到了1个SNP位点,设计合成1对错配引物。PCR后4种相应的限切酶都可以特异性地切开PCR产物,将药用野生稻或斑点野生稻与栽培稻和其它野生稻区分开来。本结果证明了dCAPS分子标记可以在口岸查验上应用。更多还原

【Abstract】 Rice is the important crops in the gramineous ones,it feeds the most of population in the world.There are abundant germplasm resource in rice,but at present,becase it lacked of corresponding inspection technology and destroyed by people, it causes our rice resources huge losses.It is necessary to protect the rice resource and diversity in order to improve the rice quality and sustainable production. For establishing a rapid and efficient rice resources molecules inspection technology system, this paper collected one hundred and five materials, Itcontains O.rufipogon, O.latiflia, O.alta, O.meyeriana.O.punctata O.officinalis, O.longistaminata, O.eichingeri,eight types of wild rices and two types of cultivated rices. It aimed to established the rice resource inspection technology system for distinguishing the species specificity of wild rice and cultivated rice.1.Using the genetic bar code technology, amplify the rice gene of trnL matK, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psb1, Adh2, Os9971,then sequencing,it conducted the system Phylogenetic evolution relation analysi by using MEGA4 for the better sequence result of trnL, rpoB, rpoC, Nadl, ndhJ, atpF-atpH, psbK-psbl and combining with the genetic sequence of genebank published information,the following conclusion (1) trnL had the more polymorphism sites,it can not only distinguish the genetic generic crops in the gramineous ones,but also locate the genomic,it divided the analytical rice material into AA,CC, DD, CCDD four chromosome type. (2) Comparing with other rice material, O.latifolia and O.alta has relative near genetic relationship. (3) atpF-atpH, rpoB, rpoC,ndhJ gene is suit to identify the above of generic crops.2. The dCAPS molecular markers system is established on the basis of the SNP, Nucleotide sequences of eight genes in O. punctata and O.officinalis were analyzed by bio-informatics software to search for SNP loci that are suitable for designing appropriate mismatch primer(s), which allow relevant enzymes to cut on after PCR. In matK gene of two wild rice species DNA, one appropriate SNP loci were found respectively, In trnL gene of O. punctata one appropriate SNP loci were found. on the basis of which four mismatch primer pairs were designed and distinguished O. punctata or O.officinalis from other O.sativa and wild rice after restriction enzyme digestion of PCR products. This result supported that dCAPS molecular markers can be used in port inspection.更多还原