[1]刘丽丽,金则新 *,李建辉,等.东南石栎ISSRPCR反应体系的优化[J].西北林学院学报,2008,23(05):65.
 LIU Li li,JIN Ze xin*,LI Jian hui,et al.Optimization of ISSRPCR Reaction System of Lithocarpus harlandii[J].JOURNAL OF NORTHWEST FORESTRY UNIVERSITY,2008,23(05):65.
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东南石栎ISSRPCR反应体系的优化()
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《西北林学院学报》[ISSN:1001-7461/CN:61-1202/S]

卷:
第23卷
期数:
2008年05期
页码:
65
栏目:
出版日期:
2008-09-30

文章信息/Info

Title:
Optimization of ISSRPCR Reaction System of Lithocarpus harlandii
作者:
刘丽丽12金则新2 *李建辉2李钧敏2
1. 北京林业大学 自然保护区学院,北京100083;2. 台州学院 生态研究所,浙江 临海 317000
Author(s):
LIU Lili12JIN Zexin2*LI Jianhui2LI Junmin2
1. College of Natural Reserves, Beijing Forestry University, Beijing 100083, China; 2. Institute of Ecology, Taizhou University, Linhai, Zhejiang 317000, China
关键词:
东南石栎ISSR优化遗传多样性
Keywords:
Lithocarpus harlandii ISSR optimization
分类号:
S792.180.4
文献标志码:
A
摘要:
为了利用ISSR分子标记进行东南石栎(Lithocarpus harlandii)种群遗传多样性分析,获得重复性和可靠性较高的ISSR带谱,有必要对其影响因素进行优化。以东南石栎基因组DNA为研究对象,测试东南石栎ISSR扩增的最适退火温度,并采用单因素试验,对影响PCR扩增效果的一些因素,如Mg2+浓度、4×dNTP浓度、模板DNA用量、引物用量、BSA浓度、Taq DNA聚合酶用量等6个因素进行筛选和优化,最终确立了可用于东南石栎ISSRPCR分析的最适宜的PCR反应条件:10 μL PCR反应体积中,1×Taq酶配套缓冲液(200 mmol·L-1 TrisHCl,pH值8.8,100 mmol·L-1 KCl,1% Triton X100),0.5U Taq DNA聚合酶,2.25 mmol·L-1 MgCl2,0.5 mmol·L-1 4×dNTP,6pmol引物,2 mg·mL-1 BSA,10 ng模板DNA。在此最适条件下,比较了降落PCR的扩增结果与最适退火温度条件下的PCR扩增结果的差异,确定了最适的ISSR扩增程序:94℃预变性5 min,94℃变性1 min,57℃退火1 min(每个循环降低0.5℃),72℃延伸1.5 min,共10个循环;94℃变性1 min,52℃退火1 min,72℃延伸1.5 min,共25个循环;72℃延伸5 min。以此条件采用12个引物对千岛湖的东南石栎居群进行扩增,共扩增出174个DNA片段,多态位点百分率为81.61%,种群内遗传多样性处于较高水平。
Abstract:
In order to establish the best ISSRPCR reaction system of Lithocarpus harlandii. The annealing temperature were determined, factors affecting the PCR amplification were selected and optimized, such as Mg2+ concentration, dNTP concentration, DNA templates dosage, primer dosage, BSA concentration, and Taq DNA polymerase dosage were selected and optimized. The results showed that the optimal ISSR reaction system for L. harlandii was as follows: 1×Taq polymerase buffer (200 mmol·L-1 TrisHCl, pH8.8, 100 mmol·L-1 KCl and 1% Triton X100), 0.5U Taq DNA polymerase, 2.25 mmol·L-1 MgCl2, 0.5 mmol·L-1 4×dNTP, 2 mg·mL-1 BSA, 10 ng DNA template, 6pmol primer in total 10 μL reaction volume. The ISSR amplification results using touchdown or universal program were compared and the suitable ISSR amplification procedure was determined: predenaturation at 94℃ for 5 min,denaturation at 94℃ for 1 min, annealing at 57℃ for 1 min(decrease 0.5℃ every cycle),elongation at 72℃ for 1.5 min,total 10 cycles; then denaturation at 94℃ for 1 min,annealing at 52℃ for 1 min,elongation at 72℃ for 1.5 min,total 25 cycles; final elongation at 72℃ for 5 min.12 random primers were selected in the ISSR amplification of Qiandaohu population of L. harlandii and 142 repetitive loci were produced. The percentage of polymorphic loci was 81.61% indicating the relatively high genetic diversity within Qiandaohu population of L. harlandi.

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备注/Memo

备注/Memo:
收稿日期:20071207修回日期:20071227 基金项目:浙江省自然科学基金项目(Y505331);浙江省科研计划资助项目(20040287)。 作者简介:刘丽丽,女,硕士研究生,主要从事植物生态学研究。 *通讯作者:金则新,男,教授,主要从事植物生态学研究。Email: jzx@tzc.edu.cn
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