Optimization of Quantitative Real-time PCR System on Amplification of Beta-glucosidase Gene Os1bglu4  

作　　者：Rouyi CHEN Jiang CHENG Changxiang ZHENG Minna PAN Mariena KETUDAT-CAIRNS 

机构地区：[1]Guizhou Institute of Upland Food Crops,Guizhou Academy of Agricultural Sciences [2]School of Biotechnology,Suranaree University of Technology

出　　处：《Agricultural Science & Technology》2014年第7期1105-1108,1218,共5页农业科学与技术（英文版）

基　　金：Supported by Guizhou International Cooperation Project on Science and Technology[(2013)7040];the 20th Project of the Joint Committee on Scientific and Technical Cooperation between the Government of the Kingdom of Thailand and the Government of the People’s Republic of China (20-606J);the Fund from Suranaree University of Technology,Thailand (SUT3-304-54-12-29)

摘　　要：[Objective] This study aimed to establish a quantitative real-time PCR （qRT-PCR） system for detecting the expression of rice beta-glucosidase gene Os1bglu4.[Method] The PCR was conducted with SYBR Green Ⅰ method,using the primers of reference gene actin or ubiquitin.[Result] Actin was more suitable to be the reference gene than ubiquitin.More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results,so,0.4 μmol/L was selected as the optimal primer concentration in this study.The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃,therefore,annealing temperature was set at 60 ℃.Compared with the reaction system of 25 μl,the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So,the 10 μl reaction system was selected,which significantly reduces the research costs for the detection of a large amount of samples in future study.[Objective] This study aimed to establish a quantitative real-time PCR(qRT-PCR) system for detecting the expression of rice beta-glucosidase gene Os1bglu4. [Method] The PCR was conducted with SYBR Green I method, using the primers of reference gene actin or ubiquitin. [Result] Actin was more suitable to be the reference gene than ubiquitin. More accurate results were obtained when the 100 ng cDNA template was added at a large volume and a lower concentration.The primer concentration in the range from 0.2 to 0.8 μmol/L we set had no significant influence on the results, so, 0.4 μmol/L was selected as the optimal primer concentration in this study. The amplification efficiency was greatly reduced when the annealing temperature was set at 64 ℃, therefore, annealing temperature was set at 60 ℃. Compared with the reaction system of 25 μl, the fluorescence intensity was significantly lower but the CT value did not change greatly in 10 μl system.So, the 10 μl reaction system was selected, which significantly reduces the research costs for the detection of a large amount of samples in future study.

关 键 词：RICE Β-GLUCOSIDASE Quantitative real-time PCR Os1bglu4 

分 类 号：Q78[生物学—分子生物学]