版纳甜龙竹DNA提取方法及AFLP反应体系建立研究

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

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。

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 OD 260 /OD280 value 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 8primer combinations,which indicates that A FLP markers are useful for genetic analysis of D.hamiltonii