[1]丁鸽,张代臻,于延球,等.中华补血草ISSR-PCR体系的构建与正交优化[J].南京师大学报(自然科学版),2012,35(03):93-97.
Ding Ge,Zhang Daizhen,Yu Yanqiu,et al.Establishment and Optimization of the Method of ISSR Fingerprinting Marker on Limonium Sinense[J].Journal of Nanjing Normal University(Natural Science Edition),2012,35(03):93-97.
点击复制
中华补血草ISSR-PCR体系的构建与正交优化(
)
《南京师大学报(自然科学版)》[ISSN:1001-4616/CN:32-1239/N]
- 卷:
-
第35卷
- 期数:
-
2012年03期
- 页码:
-
93-97
- 栏目:
-
生命科学
- 出版日期:
-
2012-09-20
文章信息/Info
- Title:
-
Establishment and Optimization of the Method of ISSR Fingerprinting Marker on Limonium Sinense
- 作者:
-
丁鸽1; 张代臻2; 于延球2; 赵玲玲1; 张蓓蓓1
-
( 1. 盐城工学院化学与生物工程学院,江苏盐城224003) ( 2. 江苏省滩涂生物资源与环境保护重点建设实验室,盐城师范学院,江苏盐城224002)
- Author(s):
-
Ding Ge1; Zhang Daizhen2; Yu Yanqiu2; Zhao Lingling1; Zhang Beibei1
-
1.Department of Chemical and Biological Engineering,Yancheng Institute of Technology,Yancheng 224003,China
-
- 关键词:
-
中华补血草; ISSR; 反应体系; 正交优化
- Keywords:
-
Limonium sinense; ISSR; reaction system; orthogonal optimization
- 分类号:
-
S567.239;R282.7
- 摘要:
-
为获得中华补血草ISSR的最佳反应体系,采用正交试验设计,对影响ISSR反应的模板含量、Mg2+浓度、dNTP浓度、Taq酶浓度、扩增程序及退火温度等多种因子进行优化筛选.通过各因子的组合研究,分析非特异性条带的产生原因并进行条件优化,建立了可用于中华补血ISSR-PCR分析的稳定可靠的反应体系:25μLPCR反应体积,10×Buffer 2.5μL,2 mmol/L MgCl2,dNTP 100μmol/L,模板DNA为20 ng,Taq聚合酶1.5 U,随机引物0.4μmol/L,退火温度52℃~58。所建立的中华补血草ISSR 反应体系具有标记位点清晰、反应系统稳定、检测多态性能力较强、重复性好等特点,可以较好地应用于中华补血草的遗传多样性及物种保护的研究.
- Abstract:
-
To obtain the optimal amplification of ISSR conditions for Limonium sinense,the effects of the concentrations of DNA templates,magesium,dNTP,Taq DNA polymerase and annealing tempreture were tested and optimized by orthogonal designed experiment. The reliable ISSR - PCR systems for L. sinense was established by analyzing the reasons for occurrence of differential bands and optimizing reaction conditions. The optimal amplification conditions for L. sinense was 25μL PCR reaction volume,10 × Buffer was 2. 5 μL,the concentration of magnesium was 2 mmol /L,dNTP was 100 μmol /L,the concentration of DNA templates was 20 ng,Taq DNA polymerase was 1. 5 U,primer was 0. 4 μmol /L. The appropriate annealing temperature was 52℃ ~ 58℃. The ISSR-PCR systems,which were established in this paper for studying L. sinense,could provide clear reliable abundant polymorphisms molecular markers and were proved suitable for studying genetic diversity and conservation of L. sinense.
参考文献/References:
[1] 陈炳华,李均,郭巧茹,等. 中华补血草多酚提取物对自由基的清除能力[J]. 植物资源与环境学报,2011,20( 3) : 36-42.
[2] Lin L C,Chou C J. Flavonoids and phenolics from Limonium sinense [J]. Planta Medica,2000,66( 4) : 382-383.
[3] 董必慧. 江苏沿海滩涂中华补血草的保护性研究[J]. 中国野生植物资源,2005,24( 6) : 28-30.
[4] Zietkiewicz E,Rafalski A,Labuda D. Genome fingerprinting by simple sequence repeats ( SSR) -anhored polymerase chain reaction amplification[J]. Genomics,1994,20( 2) : 176-193.
[5] 罗群,马丹炜,王跃华. 川乌遗传多样性的ISSR 鉴定[J]. 中草药,2006,37( 10) : 1 554-1 557.
[6] 徐红,王燕燕,魏丹红,等. 不同产地丹参药材的ISSR 分析与鉴别[J]. 中药新药与临床药理,2007,18( 6) : 454- 457.
[7] Kojoma M,Iida O,Makino Y,et al. DNA fingerprinting of Cannabis sativa using inter-simple sequence repeat ( ISSR) amplification [J]. Planta Medica,2002,68( 1) : 60-63.
[8] Jian S G,Shi S H,Zhong Y,et al. Genetic diversity among South China Heritiera littoralis detected by inter-simple sequence repeats ( ISSR) analysis[J]. Journal of Genetics and Molecular Biology,2002,13( 14) : 272-276.
[9] He J,Wang H,Li D Z,et al. Genetic diversity of Paris polyphylla var. yunnanensis,a traditional Chinese medicinal herb, detected by ISSR markers[J]. Planta Medica,2007,73( 12) : 1 316-1 321.
[10] Ge X J,Zhang L B,Yuan Y M,et al. Strong genetic differentiation of the East-Himalayan Megacodon stylophorus ( Gentianaceae) detected by inter-simple sequence repeats ( ISSR) [J]. Biodiversity and Conservation,2005,14( 4) : 849-861.
[11] Ammiraju J S S,Dholakia B B,Santra D K,et al. Identification of inter simple sequence repeat ( ISSR) markers associated with seed size in wheat[J]. Theoretical and Applied Genetics,2001,102( 5) : 726-732.
[12] Casasoli M,Mattioni C,Cherubini,et al. A genetic linkage map of European chestnu ( Castanea sativa Mill. ) based on RAPD,ISSR and isozyme markers[J]. Theoretical and Applied Genetics,2001,102( 8) : 1 190-1 199.
[13] Jin Y,Zhang W J,Fu D X,et al. Sampling strategy within a wild soybean population based on its genetic variation detected by ISSR markers[J]. Acta Botanica Sinica,2003,45( 8) : 995-1 002.
[14] Li H S,Chen G Z. Genetic diversity of Sonneratia alba in China detected by inter-simple sequence repeats ( ISSR) analysis [J]. Acta Botanica Sinica,2004,46( 5) : 515-521.[15] Dieffenbach C W,Dveksler G S. PCR 技术实验指南[M]. 北京: 科学出版社, 2000.
[16] 王培训,周联,赖小平. 分子生物学技术与中药鉴别[M]. 广州: 世界图书出版社,2002.
[17] 沈洁,丁小余,丁鸽,等. 铁皮石斛居群差异的研究Ⅱ-ISSR 指纹标记方法的建立与优化[J]. 中国中药杂志, 2006, 31( 4) : 291-294.
[18] 贺佳,丁小余,褚必海,等. 泽泻ISSR 反应体系的建立与优化[J]. 南京师大学报: 自然科学版,2006, 29( 3) : 86-90.
备注/Memo
- 备注/Memo:
-
基金项目: 国家自然科学基金( 31000142) 、江苏省自然科学基金( BK2011421) 、盐城工学院科研项目基金、盐城工学院博士启动基金 ( XKR2010003) .
通讯联系人: 丁鸽,副教授,研究方向: 药用植物资源多样性及成分. E-mail: dingzyc@ yahoo. com. cn
更新日期/Last Update:
2013-03-11