摘要
【目的】建立适合海岛棉组织培养过程中差异基因表达研究的DDRT-PCR体系。【方法】试验选取海岛棉体胚发育过程中的胚性愈伤组织,无菌苗下胚轴为对照。对引物浓度、dNTP浓度、酶浓度和退火温度等影响差异显示PCR的关键因素进行单因素优化实验。【结果】确定了海岛棉组织培养差异显示PCR体系为(10μL):cDNA 2μL;dNTP 200μmol/L;上游引物0.4μL(10μM),下游引物0.5μL(10μM);Taq酶0.5 U,Buffer1.0μL。前5个循环退火温度为50℃,后30个循环退火温度为57.1℃。【结论】用该体系扩增,PCR产物特异性高,条带清晰,背景污染少。
【Objective】To construct a DDRT-PCR system for the research of the differential gene expression of Gossypium barbadense tissues in culture.【Method】The experiment used the embryonic callus tissue of the growing Gossypium barbadense as materials,and selected the aseptlic seedling as control to research conditions of the DDRT-PCR,optimizing the critical factors such as the concentration of primers,dNTP and enzyme and annealing temperature.【Results】Confirmed the DDRT-PCR system of sea-island cotton tissue culture is 2 μL cDNA,200 μmol/L dNTP,0.4 μL（10 uM） upper primer,0.5 μL lower primer,0.5U Taq enzyme,and 1.0 μL buffer.The annealing temperature of the former 5 cycle is 50℃,the latter 30 cycle is 57.1℃.【Conclusion】Using this system can make a specific and clear band PCR products with little background disturbers.
出处
《新疆农业科学》
CAS
CSCD
北大核心
2010年第9期1728-1732,共5页
Xinjiang Agricultural Sciences
基金
新疆维吾尔自治区科技厅高新技术项目(20080101)
新疆维吾尔自治区科技厅重大项目(200731132-4)
国家转基因专项(2008ZX08005-004)
关键词
海岛棉
差异显示PCR
体系建立
Gossypium barbadense
mRNA differential display PCR
system construction
