论文标题:分子标记技术在泽泻上的应用
The Application of Molecular Marking Technology on Alisma Orientale
论文作者 陈志彤
论文导师 肖华山；郑伟文,论文学位 硕士,论文专业 植物学
论文单位 福建师范大学,点击次数 106,论文页数 60页File Size3571k
2004-04-01论文网 http://www.lw23.com/lunwen_430504272/ 泽泻,RAPD,RFLP,克隆,测序
Alisma orientale,RAPD,RFLP,cloning sequencing
为了有效地区别各产地泽泻品种，为泽泻类道地药材的栽培、繁育和质量评价提供分子水平的依据，本文对采自三个产地(福建、江西、四川)的泽泻进行分子标记，包括RAPD、18S-26S rRNA-RFLP、18S-26S rRNA基因及其ITS片断的扩增、克隆及测序。①对RAPD的各项参数进行了一系列的比较和探索，包括模板DNA的提取方法、Taq酶的用量、dNTP的浓度等条件的优化，建立了优化反应体系。并筛选了73个随机引物，结果表明福建与江西产的泽泻为同一类，与四川产的泽泻有所区别。②由于不能区分福建的两个泽泻品种(高叶泽泻、矮叶泽泻)，故欲通过对泽泻的18S-26S rRNA基因及其ITS片断进行扩增，然后用RFLP技术进一步分析，筛选了13种限制性内切酶，结果发现内切酶AluⅠ可以较好地区分出福建、江西与四川产地泽泻的区别，与RAPD结果一致，但仍未能区分出福建高叶泽泻与矮叶泽泻。③对泽泻18S-26S rRNA基因及其ITS扩增片断在大肠杆菌里进行克隆，并对其扩增片段进行测序，测序结果与RAPD、RFLP结果一致，综合目前的分子实验结果尚不能区分开福建高叶与福建矮叶泽泻的区别。所测的福建泽泻Alisma orientale、川泽泻Alisma plantago-aquatica的18S-26S rRNA基因及其ITS片断序列均已获得Genbank数据库登录号，分别为AY519469、AY588940。
In order to identify the samples of Alisma orientale(Sam.) Juzep. from different areas in Fujian, Sichuan, and Jiangxi provinces effectively, make a technological base at molecular level for these Chinese herbs"growth, breeding and quality evaluation, this paper were mainly used molecular marking technique to study them including RAPD, RFLP, cloning and sequencing. First, many parameters of RAPD technique was studied by a series of experiments, including DNA extraction, Optimization PCR amplification by comparing a series of annealing temperatures, template concentrations, amounts of Taq DNA polymerase and so on, selection of 73 random primers, the result showed that the samples were classified into two groups, one was Alisma orientale from Fujian and Jiangxi provinces, the other was from Sichuan province, its botanical origin was Alisma plantago-aquatica. Second, two types of Alisma orientale from Fujian(tall-leaf Alisma orientale and short-leaf Alisma orientale ) still could not be identified by RAPD technique, so RFLP was applied on the samples. These samples" 18S-26S rRNA and its ITS fragments were amplified and digested with thirteen kinds of endonuclease. The result is the same as RAPD when using endonuclease Alu I on the samples. Third, 18S-26S rRNA and its ITS fragments were ligated into pMD 18-T vector and transformed in Escherichia coli, and then sequenced. The DNA sequence results were also the same as RAPD and RFLP results. So based on these experiments, we still could not identify the difference of tall-leaf Alisma orientale, short-leaf Alisma orientale and Alisma orientale from Jiangxi province, but they were different with Alisma plantago-aquatica from Sichuan province. The 18S-26S rRNA and its ITS sequence of Alisma orientale Alisma plantago-aquatica had get the accession number AY519469 AY588940 of Genbank database.
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