摘要：地黄(Rehmannia glutinosa)为玄参科地黄属多年生草本植物，广泛分布于中国北方，其地下块根可入药，为我国一著名传统中药材。本文就地黄DNA提取方法的优化进行了研究。采用改良的CTAB法，对四个参数包括：去糖与否、水浴时间、叶片重量、取材部位等条件进行优化组合配置。实验结果表明：以不同生境条件下的野生地黄作为实验材料，其优化组合相似，无明显差异。以生长在阴坡生境下的地黄叶片为例，其叶片含腺毛少，叶片上的紫色斑点少，其最适条件组合为叶片鲜重0.4克，水浴时间2小时，蛋白质抽提2次，加醋酸钠沉淀，提取得到的DNA的纯度及浓度均较高，完整度好。通过琼脂糖电泳可观测到清晰单一的条带，进一步的浓度检测结果表明其A260/A280在1.8~2.0左右，DNA浓度在4000~6400ng/μL。本文的实验结果为地黄在分子生物学方面的进一步研究提供了数据参考。43095

毕业论文关键词：地黄；基因组DNA提取；改良的CTAB法

Optimization of Methods for Genomic DNA Extraction on Rehmannia glutinosa

Abstract: Rehmannia glutinosa (Scrophulariaceae) is a perennial herb, commonly distributed in North China. Its underground roots can be used as a traditional Chinese medicine. In this study, based on the cetyltrimethyl ammonium bromide (CTAB) method, we optimized the extraction methods for genomic DNA of R. glutinosa ,including four different parameters: adding the remove sugar buffer or not, different time for 65°C incubation, the weight of fresh sample and the different tissues . Our results indicated that: there was no significant difference between different habitats of Rehmannia glutinosa at the protocol for optimal quality and quantity genomic DNA isolation . For the inpiduals from the shade habitat,they have less glandular hairs, fewer purple spots on the leaves. The optimum protocol for the shade inpiduals was as follows: fresh leaf weight of 0.4 g, 65°C incubation for 2 hours, chloroform extraction to get rid of proteins twice, addition of NaAc to assist DNA precipitation. Through agarose gel electrophoresis, a single robost clear band was observed. Further concentration detection showed that the value of  A260 / A280 was 1.8 to 2.0,the range of the concentration was 4000~6400ng/μL. The results of this research provided the basic data for R. glutinosa at further research in molecular biology.

Key words:  Rehmannia glutinosa; Genomic DNA; Improved CTAB method  

目    录

摘  要 1

引言 2

1．基本原理 3

2. 材料与方法 3

2.1 实验材料 3

2.2 DNA提取 4

2.3 实验处理 4

2.4 检测方法 5

2.4.1 琼脂糖凝胶电泳检测 5

2.4.2 紫外分光光度计检测 5

3. 结果与分析 5

3.1 琼脂糖凝胶电泳结果 5

3.1.1 不同水浴时间的电泳结果 5

3.1.2 不同叶片重量的电泳结果 5

3.1.3腺毛多少及是否去糖等条件组合的电泳结果 6

3.2 紫外分光光度计检测结果 6

4. 结论与讨论 8

4.1 结论 8

4.2 讨论 9