干旱区生态环境知识资源中心(Arid): 荒漠刺叶墙藓DNA的提取及PCR扩增反应条件

Arid

	荒漠刺叶墙藓DNA的提取及PCR扩增反应条件
其他题名	DNA Extraction, Optimization of PCR Amplification Conditions and Selection of Primers of Tortula desertorum in Biotic Crust in the Gurbantonggut Desert
	王红玲; 张元明; 吴楠; 陈荣毅; 聂华丽; 张静
ISSN	1001-4675
出版年	2008
卷号	25 期号:1 页码:98-106
中文摘要	采用改进CTAB法、NaOH法和直接PCR法（direct PCR amplification）提取藓类生物结皮中刺叶墙藓（Tortula desertorum Broth．）基因组DNA，比较其分离效果以及ISSR扩增效果。结果表明：改进CTAB法提取的基因组DNA质量好、纯度高，ISSR扩增反应效果好。直接PCR法一旦体系建立，则是最简易快速、经济高效的模板制备方法，适于植株矮小、生物量很小的荒漠藓类植物个体模板DNA的制备。利用改进CTAB法获得的基因组DNA作为模板。对ISSR反应组分浓度及反应程序中的一些重要参数进行摸索和优化实验，建立了刺叶墙藓最优ISSR反应体系。反应总体积为20muL，各反应组分终浓度为：2muL 10*Buffer，2mM Mg~(2＋)，0．1mM dNTPs，0．2muM引物，10-40ng模板DNA，1U Taq酶。扩增程序为：94℃ 4min，1个循环；94℃45s，退火温度（Tm）45s，72℃1min，35个循环；72℃7min，1个循环；4℃保存。刺叶墙藓是组成古尔班通古特沙漠藓类生物结皮的优势种，研究结果为进一步开展刺叶墙藓遗传多样性的研究奠定了基础。
英文摘要	In this study, the DNA of Tortula desertorum Broth. in biotic crust in the Gurbantonggut Desert is extracted using the methods of modified CTAB, NaOH extraction and direct amplification, and the isolation and ISSR amplification effects of these methods are compared. The results show that the content and purity of genomic DNA extracted with the modified CTAB method is very high, and the DNA can be successfully used in ISSR amplification. Once the PCR amplification system is developed, the direct PCR amplification is the rapidest and simplest, and the cost is the lowest in preparing plant sample for PCR, especially the method is applicable to extract the DNA from the short desert mosses with very low biomass. Based on the genomic DNA extracted from Tortula desertorum with the modified CTAB method, the main factors affecting the result of ISSR amplification are tested and compared. An optimal ISSR reaction system applicable for extracting the DNA of Tortula desertorum is developed. The total 20muL volume is consisted of 2 muL 10 * buffer, 2 mM Mg~(2＋), 0. 1mM dNTPs, 0.2 muM primer, 10 - 40 ng template DNA and 1 U Taq polymerase. The amplification profile is consisted of one cycle of 4 minutes at temperature of 94 ℃, 35 cycles of 45 s at temperature of 94 ℃, 45 s at anneal temperature and 1 minute at temperature of 72 ℃, and one cycle with a final extension step of 7 minutes at temperature of 72 ℃. Tortula desertorum is the dominant species in biotic soil crust in the Gurbantonggut Desert, and the research achievement can be regarded as the base of further studying the genetic diversity of Tortula desertorum.
中文关键词	刺叶墙藓 ; DNA提取 ; ISSR扩增 ; 藓类生物结皮 ; 古尔班通古特沙漠
英文关键词	Tortula desertorum Broth. DNA extraction ISSR alnplification moss biotic soil crust Gurbantonggut Desert
语种	中文
国家	中国
收录类别	CSCD
WOS类目	PLANT SCIENCES
WOS研究方向	Plant Sciences
CSCD记录号	CSCD:3255526
来源机构	中国科学院新疆生态与地理研究所
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/222310
作者单位	中国科学院新疆生态与地理研究所, 乌鲁木齐, 新疆 830011, 中国
推荐引用方式 GB/T 7714	王红玲,张元明,吴楠,等. 荒漠刺叶墙藓DNA的提取及PCR扩增反应条件[J]. 中国科学院新疆生态与地理研究所,2008,25(1):98-106.
APA	王红玲,张元明,吴楠,陈荣毅,聂华丽,&张静.(2008).荒漠刺叶墙藓DNA的提取及PCR扩增反应条件.,25(1),98-106.
MLA	王红玲,et al."荒漠刺叶墙藓DNA的提取及PCR扩增反应条件".25.1(2008):98-106.