版纳甜龙竹DNA提取方法及AFLP反应体系建立研究

doi:10.7668/hbnxb.20102.S1.009

华北农学报 ›› 2010, Vol. 25 ›› Issue (S1): 32-37. doi: 10.7668/hbnxb.20102.S1.009

版纳甜龙竹DNA提取方法及AFLP反应体系建立研究

杨清^1,2, 苏光荣³, 韩蕾⁴, 王正良¹, 孙启祥⁴, 彭镇华⁴

1. 中国科学院西双版纳热带植物园, 云南勐腊 666303;
2. 国际竹藤网络中心, 中国林业科学研究院研究生院, 北京100091;
3. 云南省景洪市林业局, 云南景洪 666100;
4. 中国林业科学研究院林研所, 北京 100091

收稿日期:2010-04-04 出版日期:2010-12-30
通讯作者: 孙启祥(1967-),男,安微巢湖人,研究员,博士,主要从事森林培育与生物多样性保护研究
作者简介:杨清(1969-),男,重庆忠县人,副研究员,博士,主要从事植物遗传多样性研究与森林培育工作
基金资助:
中科院方向知识创新项目“热带优质笋材竹种筛选评价与开发利用”;国家科技部基础条件平台项目(2004DKA30400-05-01-02)

Study on Genomic DNA Extraction Method and AFLP Amplification Reaction System of D. hamiltonii Nees et Arn. ex Munro

YANG Qing^1,2, SU Guang-rong³, HAN Lei⁴, WANG Zheng-liang¹, SUN Qi-xiang⁴, PENG Zhen-hua⁴

1. Xishuangbanna Tropical Botanic Gardens,Chinese Academy of Science, Menglun 666303, China;
2. Graduate School of International Centre for Bamboo and Rattan&Chinese Academy of Forestry, Beijing 100091, China;
3. Forestry Department of Jinghong City in Xishuangbanna, Jinghong 666100, China;
4. Forestry Institute of Chinese Academy of Forestry, Beijing 100091, China

Received:2010-04-04 Published:2010-12-30

摘要/Abstract

摘要： 用改进的CTAB法,在提取液中加入2%PVP(V/V)和2%β-巯基乙醇,提取到了高质量的基因组DNA,OD₂₆₀/OD₂₈₀在1.7～1.9之间,蛋白质、多酚类、色素、RNA等去除较彻底,适于AFLP分析。通过对版纳甜龙竹DNA提取、酶切连接、预扩增、选择性扩增等试验过程中各关键因素的比较研究,建立了优化的AFLP分子标记体系,得到了清晰的AFLP银染指纹图谱,试验结果重复性好。从64对MseI和PstI引物中筛选出8对扩增效果好的引物,利用筛选出8对引物对30份材料进行分析,共扩增出256条多态性带,获得32490个扩增位点,有13289个位点具有多态性,平均每对引物获得4061.25个扩增位点,其中具有多态性的位点为1661.125个,平均多态检出率为40.90%,8对引物组合对版纳甜龙竹30个个体的区分率达到100%。表明AFLP用于版纳甜龙竹种内不同材料间的遗传变异性分析是可行的。

关键词: 版纳甜龙竹, DNA提取, AFLP, 影响因素

Abstract: The study on extraction of DNA with improved CTAB (cetyl-trimethyl-ammonium bromide) method from Dendrocalamus hamiltonii Nees et Arn. ex Munro. It showed that CTAB buffer supplemented with 2% PVP (poly vinyl pyrrolidone) and 2% β-Mercaptoethanal was suitable for the genomic DNA extraction of D. hamiltonii Nees et Arn. ex Munro,with OD₂₆₀ /OD₂₈0value of 1. 7－1. 9,representing high purity of the extracted DNA and the complete elimination of protein,phenol,pigment and RNA. Thus the improved CTAB method was approved suitable for further AFLP analysis of D. hamiltonii. Some key factors affecting DNA extraction,restriction-ligase reaction,preamplification and amplification processes were studied and an optimized AFLP reaction system of D. hamiltonii was established. With this AFLP reaction system,clear DNA electrophoresis fingerprints of D. hamiltonii were obtained, and the reproducibility was high. Eight primer combinations were filtrated from sixty and four pair with Mse I and Pst I,8 primer combinations with stable amplification and rich polymorphism for AFLP were screened,256 DNA bands were amplified by eight pairs of AFLP primers and 40. 90% polymorphic bands per pair of primer were produced on average,which had 32 490 distinct and strong amplification bands with a bright background and 13 289 of them were polymorphic bands from 30 individuals of D. hamiltonii,we can entirely distinguish 30 individuals by 8 primer combinations,which indicates that A FLP markers are useful for genetic analysis of D. hamiltonii.

Key words: Dendrocalamus hamiltonii Nees et Arn. ex Munro, DNA extraction, AFLP, Influential factor

中图分类号:

S795.03

杨清, 苏光荣, 韩蕾, 王正良, 孙启祥, 彭镇华. 版纳甜龙竹DNA提取方法及AFLP反应体系建立研究[J]. 华北农学报, 2010, 25(S1): 32-37. doi: 10.7668/hbnxb.20102.S1.009.

YANG Qing, SU Guang-rong, HAN Lei, WANG Zheng-liang, SUN Qi-xiang, PENG Zhen-hua. Study on Genomic DNA Extraction Method and AFLP Amplification Reaction System of D. hamiltonii Nees et Arn. ex Munro[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(S1): 32-37. doi: 10.7668/hbnxb.20102.S1.009.

http://www.hbnxb.net/CN/Y2010/V25/IS1/32

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