论文标题:香榧主要栽培品种的RAPD分子鉴别
Studies on the ingredients of the mellus Doumeri Leaf and its health care function
论文作者 张党权
论文导师 谭晓风,论文学位 硕士,论文专业 森林培育
论文单位 中南林学院,点击次数 80,论文页数 49页File Size3388k
2001-05-01论文网 http://www.lw23.com/lunwen_768005667/ 香榧;RAPD标记;DNA抽提;分子鉴别;分子分类
Torreya grandis, RAPD marker, DNA extraction, molecular identification, molecular classification
香榧（Torreya grandis）为我国特有的珍贵多用途经济树种，其种子为著名的干果，全身都是宝，经济价值非常高。香榧是我国近年来发展最快、经济效益最好的经济林树种之一，优良无性系品种越来越多。但由于良种枝条和苗木的缺乏，一些不法之徒以次充好，以假冒真的事件在各地时有发生，因此鉴别香榧优良品种已成为当前香榧生产和良种化过程中急需解决的迫切问题。香榧的品种分类研究仍限于形态学、解剖学的方法，有关香榧在DNA水平上的分类研究尚未见报道。本实验试图利用RAPD技术对香榧主要栽培品种进行系统聚类分析，并进行品种种类的划分和分子鉴别，为更加深入地研究这一珍贵树种发挥其潜在的巨大经济价值提供依据。 本实验利用香榧种子胚乳DNA来进行RAPD分析。共有8个品种，分别为：茄榧、旋纹榧、大圆榧、小圆榧、细榧、米榧、芝麻榧、冲杠榧。采用改良的CTAB法对香榧种子胚乳DNA进行抽提。将抽提出来的DNA溶于TE缓冲液中，利用Du-640核酸蛋白分析仪测定DNA的浓度。结果表明，所抽提的香榧种子胚乳DNA样品中，DNA浓度较高，但仍含有较多的蛋白质等杂质，而且DNA的纯度不高，但由于RAPD分析对模板DNA的纯度要求不高，所抽提的DNA样品完全能满足RAPD分析的需要。 为了提高RAPD分析的重复性，在实验过程中对RAPD分析的各个步骤都进行了反复的摸索，特别是对RAPD的反应体系和循环条件进行了优化，最后得到了较优的适合于香榧种子胚乳DNA进行RAPD分析的反应条件。其中RAPD反应体系为：10ng/μl DNA template 2.0μl；10x buffer 1.5μl；2mM dNTPs 1.8μl；25mM MgCl_2 1.44μl；10μM primer 1.26μl；5U Tag polymerase0.2μl；ddH_2O 6.8μl；total 15μl。RAPD-PCR循环条件为：95℃变性3min，一个循环；94℃变性30s，36℃退火30s，72℃延伸60s，35个循环；72℃延伸5min，反应终止。 从520种随机引物种筛选出23种引物，对香榧8个主要栽培品种进行RAPD分析，产生了80个标记位点，并在此基础上得到了香榧8个主要栽培品种的标记基因型，并以此标记基因型进行分子鉴别。将香榧的标记基因型剖分成0、1、2型三组数据，采用模糊聚类分析软件进行分析，对香榧8个主要栽培品种进行分子分类，并将其分为两大类。
Torreya grandis is the special and precious economic tree with many uses in our country. Its whole body is just a gem and its seed is a well-known nut with a high economic value. In the recent years more and more improved breeds of Torreya grandis have been cultivated with the rapid productive development as one of trees with high economic benefit. But because of the lack of branches and nursery stocks of improved varieties, some cheats often happened from lawless persons by using the sham as the true. So at present the urgent problem during the course of producing and improving breeds is how to identify the ? improved breeds of Torreya grandis. Because the research on the breed classification of Torreya grandis is still limited to the research methods of morphology and anatomy, so it is very hard to find any article about the classification research at the level of DNA. In these ? experiments we try to perform the systematical analysis of assembling classes about the main cultivated breeds of Torreya grandis, and try to divide and identify the varieties and classes by using RAPD technology. The aim of this research is to provide data for the further deepen study of this precious tree species for the potential economic value. In this research the material for the analysis of RAPD is the endosperm DNA of Torreya grandis seed. there are eight breeds: qiefei, xuanwenfei, dayuanfei, xiaoyuanfei, xifei, mifei, zhimafei, chonggangfei. The DNA samples of endosperm of Torreya grandis seed which are extracted by using improved CTAB method are dissolved into TE buffer liquid for the purpose of determining its DNA density by using Du-640 nucleic acid and protein analytical instrument. The result indicates that the DNA samples content a high density DNA and considerable impurities such as protein, and its purity is not high. But the DNA samples can totally meet the need of RAPD analysis because RAPD doesn抰 need a high purity template DNA. For the purpose of improving the repeativeness of RAPD we probe for all procedures of RAPD analysis, especially for the reaction system and circular condition of RAPD during the course of the experiments. At last the optimal reaction conditions are established for the RAPD analysis of endosperm DNA of Torreya grandis. The reaction system of RAPD is: lOng/jil DNA template 2.0~il; lOx buffer l.Sixl; 2mM dNTPs 1.8j1l; 25mM MgC12 l.44iil; lOj.tM primer 1.26p1; 5U Tag polymerase 0.2pJ; ddH2O 6.8p1; total ISp]. the circular condition ofRAPD is: 9500, 3mm; one circle; 9400, 30s, 3600, 30s, 7200, 60s, 35 circles; 72 ~C 5mm. The DAN samples of eight man cultivated breeds of Torreya grandis are analyzed with RAPD, and 80 marker sites are found by using 23 primers that are screened from 520 random primers. The marker genotype of eight man cultivated breeds of Torreya grandis is formed and the molecular identification system is established on the base of it. Eight man cultivated breeds of Torreya grandis are molecularly classified into two classes after the marker genotype is transformed into data of 0-1 -2 kind which is analyzed with a assembling class software of 0-1-2 kind.
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