[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for furthe...[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.展开更多
Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat...Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.展开更多
LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits nor...LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.展开更多
[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connec...[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connected into bait vector pGBKTT. The recombined vector was transformed into yeast competent ceils AH109 and carried out self-activated detection and toxicity detection in the au.xotrephic medium. [ Result] Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was suecessfully constructed with correct reading frame. Self-activated activity and toxicity detection results showed that bait vector had no self- activated activity on yeast strain AH109, which also had no toxicity on yeast. [Condusion] Bait vector successfully constructed could be used in yeast two-hybrid system, which laid the foundation for screening of cDNA library in the next step.展开更多
文摘[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.
基金the National Natural Science Foundation of China (Grant No. 3967017330170441) "863"Project (Grant No. 2001AA221161)+1 种基金Beijing Natural Science Foundation (7002026) High Education Science Research Foundation of China (20010023024).
文摘Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.
文摘LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.
基金Supported by Natural Science Foundation of Beijing (6012017)Dr. Funding of Xinjiang Production and Construction Corps~~
文摘[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connected into bait vector pGBKTT. The recombined vector was transformed into yeast competent ceils AH109 and carried out self-activated detection and toxicity detection in the au.xotrephic medium. [ Result] Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was suecessfully constructed with correct reading frame. Self-activated activity and toxicity detection results showed that bait vector had no self- activated activity on yeast strain AH109, which also had no toxicity on yeast. [Condusion] Bait vector successfully constructed could be used in yeast two-hybrid system, which laid the foundation for screening of cDNA library in the next step.
