摘要
cDNA-SCoT差异显示分析是应用于植物基因差异表达研究的一项新技术。本研究以甘蔗品种桂糖11号根尖为材料,提取总RNA并反转录第1链cDNA,对影响甘蔗cDNA-SCoT差异显示PCR反应的cDNA模板、SCoT引物、dNTPs、Taq DNA聚合酶和Mg2+等因子进行优化,建立甘蔗cDNA-SCoT反应体系,同时应用优化后反应体系分离25%PEG6000胁迫诱导甘蔗根尖差异表达基因片段。结果表明:各项因子均对PCR扩增效果存在影响,20μL反应体系中各因子的最佳含量如下:cDNA 80 ng、引物浓度1.0μmol/L、Taq DNA聚合酶2.0 U、dNTPs浓度200μmol/L、Mg2+浓度1.8 mmol/L。研究结果还显示,SCoT引物扩增多态性丰富,平均每条引物扩增条带30条,多态性条带比率为46.9%,6条引物分离得到PEG诱导甘蔗根尖特异表达片段45条,表明本研究所建立的甘蔗cDNA-SCoT反应体系具有操作简便、结果稳定、重复性好、成本低、多态性丰富等优点,可为甘蔗基因差异表达研究提供新的技术支持。
Gene differential expression analysis by cDNA-SCoT is a newly developed technique in plants. In this study, the root tip of sugarcane variety GTll was used as the experiment material. We isolated the total RNA of sugarcane root tip and it was reversely transcripted into the first strand cDNA. And then, we optimized SCoT-PCR amplification system of sugarcane in five factors such as template cDNA, SCoT primers, Taq DNA polymerase, dNTPs and Mg2＋ to establish an optimal reaction system of sugarcane, which was used to isolate differentially expressed genes in sugarcane root tip induced by 25% PEG6000. The results showed that all of the five factors affected the PCR amplification and the optimal 20 p^L PCR reaction mixtures in sugarcane was template cDNA 80 ng, SCoT primers 1.0 p^mol/L, Taq DNA polymerase 2.0 U, dNTPs 200 ~mol/L and Mg2＋ 1.8 mmol/L. The amplification results of SCoT primers indicated higher genetic diversity at the polymorphic rate of 46.9%, and the average bands of each primer were 30 bands. What should be stressed is that we obtained a total of 45 differentially expressed bands in sugarcane root tip induced by 25% PEG. The cDNA-ScoT PCR amplification system established in this paper has several advantages： simplicity, stabilization, highly reproducibility, inexpensiveness and rich polymorphism, which would be a new technique useful for sugarcane as differential gene expression analysis.
出处
《热带作物学报》
CSCD
北大核心
2013年第5期892-898,共7页
Chinese Journal of Tropical Crops
基金
863计划课题(No.2013AA102604)
国家国际合作项目(No.0S2013ZR0130)
国家科技支撑计划项目子课题(No.2012BAD40B04)
广西自然科学基金创新团队项目(No.2011GXNSFF018002)
广西农科院团队项目(No.桂农科2011YT01)
