【作者】 巩艳红；

【导师】 刘军；

【作者基本信息】 四川农业大学 ， 森林培育学， 2003， 硕士

【摘要】 本试验以毛豹皮樟的叶片为材料，通过对不同的DNA提取方法进行试验，寻找最佳的毛豹皮樟DNA提取方法；优化扩增程序和反应条件，建立起毛豹皮樟的RAPD技术体系；并将该技术体系应用于80个毛豹皮樟叶片的RAPD扩增中。结果表明： 一、使用改良SDS提取法有效地解决了毛豹皮樟基因组总DNA提取中的褐变问题，同时所提取的DNA均呈白色絮状，A₂₆₀/A₂₈₀值都在1.8以上，从检测所得产率和纯度可知该DNA模板完全可以用于RAPD分析；另外，该方法还具有成本低、操作简单、省时等特点。 二、用CTAB法提取毛豹皮樟叶片的DNA时，所得DNA量很少，而且模板中含色素、蛋白质等杂质很多，在后期进行RAPD扩增时，杂质的影响导致扩增不稳定或者扩增失败，表明CTAB提取法对于含酚类等次生物质较多的物种并不适用。采用常规的SDS法所得的DNA量很少，而且模板中含色素、蛋白质等杂质很多，在后期进行RAPD扩增时，杂质的影响导致扩增不稳定或者扩增失败。 三、扩增程序采用：预变性温度为94℃1min，变性温度为94℃1min，复性温度为34℃1min，72℃延伸2min，共45个循环，最后72℃延伸10min，同时反应体系（20μl反应体系）采用：dNTPs0.4μl（各含1Nm/μl），10×PCR2μl，MgCl₂μl，Taq酶0.3μl（0.5u/μl），H₂O11.3μl，DNA模板2μl（40ng），Primer2μl（0.8Pm/μl），能够得到清晰稳定的扩增带谱。 四、将所建立的RAPD技术体系应用于20个毛豹皮樟叶片DNA的RAPD扩增中，筛选出16个引物，所得的谱带清晰，多态性高达46.8％，反映了各试材之间在DNA序列上存在相当大的差异，可用于毛豹皮樟的种质资源鉴定分析。 五、用筛选出的部分随机引物对80个毛豹皮樟的DNA模板进行RAPD扩增，得到了稳定的RAPD带谱。这些特征引物对各种材料扩增所得到的特征谱带反映了基因组总DNA碱基序列的特异性，可用于聚类分析。更多还原

【Abstract】 Using the leaves of Litsea coreana LevL var. lanuginosa as materials, different waysof isolating genomic DNA were tested to find the best one. At the same time, the optimal Random Amplified Polymorphic DNA (RAPD) techniques for Litsea coreana LevL var. lanuginosa (Migo) Yang et P.H.Huang were established and applied to 80 leaves. The results were as follows:1) Using improved SDS method was an economic, easy and fast way to isolate genomic DNA. It could obtain qualified DNA from plants containing high polyphenols. The DNA was qualified for RAPD analysis.2) Using CTAB method and SDS method could isolate only a little amount of DNA, farther more, the DNA contains secondary substances such as pigment and protein. As a result, the amplification was instable or failed.3) The PCR products were stable when the PCR was 45 cycles of predenature at 95 for 1 minute, denature 94 1 minute, annealing 34 1 minute, extension 72 3 minutes, and when the amplification reaction solution (20ul) was consisted of dNTPs 0.4 l, 10 PCR2 l, MgCl22ul, Taq DNA polymerase 0.3 l(0.5u/ l), H2O 11.3 l , template DNA 2ul(40ng), Primer 2 l(0.8Pm/ l).4) The results also showed that Litsea coreana LevL var. lanuginosa possessedmuch higher genetic diversity and the genetic diversity degree was 46.8%. The specifkpresence or absence ef amplified bands of different materials from different primers,which revealed the speciality in sequences of genomic DNA, could be used for identifying variety of Litsea coreana LevL var. lanuginosa.5) Eighty template DNAs were tested with RAPD markers using 16 arbitrary 10-mer primers selected from 38 primers.更多还原