[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, usin...[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.展开更多
In order to analyze the Os1bglu4 phenotype,the inducible promoter of the transgenic rice which knock-down the Os1bglu4 expression was assessed.The result showed that 30μM dexamethasone(DEX)had the stronger induction ...In order to analyze the Os1bglu4 phenotype,the inducible promoter of the transgenic rice which knock-down the Os1bglu4 expression was assessed.The result showed that 30μM dexamethasone(DEX)had the stronger induction effect than10μM DEX byβ-Glucuronidase(GUS)staining.qRT-PCR further verified the Os1bglu4 gene deletion.The effect of DEX and its solvent absolute ethanol on seed development was measured,and no significant effect was observed.The conclusion is that final concentration of DEX at 30μM is suitable for pOp6 promoter induction.展开更多
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)
文摘[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.
基金Supported by Guizhou International Cooperation Project on Science and Technology[No.QiankehewaiG(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(No.20-606J)China.Suranaree University of Technology grant number SUT3-304-54-12-29,Thailand
文摘In order to analyze the Os1bglu4 phenotype,the inducible promoter of the transgenic rice which knock-down the Os1bglu4 expression was assessed.The result showed that 30μM dexamethasone(DEX)had the stronger induction effect than10μM DEX byβ-Glucuronidase(GUS)staining.qRT-PCR further verified the Os1bglu4 gene deletion.The effect of DEX and its solvent absolute ethanol on seed development was measured,and no significant effect was observed.The conclusion is that final concentration of DEX at 30μM is suitable for pOp6 promoter induction.
