Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction （RT-PCR）, using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open rea...Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction （RT-PCR）, using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open reading frame （ORF）, which encodes a 444 amino acid protein. Semi-quantitative RT-PCR analysis of GATA-3 mRNA expression was done using the total RNAs from different normal tissues of a large white pig. The GATA-binding family of transcription factors comprised of a subgroup of DNA-binding proteins that both bound the consensus GATA motif and contained the class Ⅳ zinc finger motif. The molecular evolution tree was constructed based on the GATA-3 amino acid sequence and class Ⅳ zinc finger motif using mega 3.1. Phylogeny analysis of GATA factors isolated from vertebrates suggested that the six distinct vertebrate GATAs had descended from a common ancestral sequence, and the topology also suggested multiple modes of evolution including gene duplication and class Ⅳ zinc finger motif recombination. These data helped the authors in illuminating the pathways of divergent and convergent evolution of the GATA-binding family of transcription factors.展开更多
[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,us...[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
基金This work was supported by the Heilongjiang Province Tackle Project (No. GB01B104).
文摘Pig GATA-3 cDNA was obtained by reverse transcription polymerase chain reaction （RT-PCR）, using in silico cloning strategy based on pig dbEST. The length of pig GATA-3 cDNA is 1,760 bp containing a 1,335 bp open reading frame （ORF）, which encodes a 444 amino acid protein. Semi-quantitative RT-PCR analysis of GATA-3 mRNA expression was done using the total RNAs from different normal tissues of a large white pig. The GATA-binding family of transcription factors comprised of a subgroup of DNA-binding proteins that both bound the consensus GATA motif and contained the class Ⅳ zinc finger motif. The molecular evolution tree was constructed based on the GATA-3 amino acid sequence and class Ⅳ zinc finger motif using mega 3.1. Phylogeny analysis of GATA factors isolated from vertebrates suggested that the six distinct vertebrate GATAs had descended from a common ancestral sequence, and the topology also suggested multiple modes of evolution including gene duplication and class Ⅳ zinc finger motif recombination. These data helped the authors in illuminating the pathways of divergent and convergent evolution of the GATA-binding family of transcription factors.
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.
