With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to ...With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.展开更多
Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol （TG） leads to fatty liver disease encompassi...Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol （TG） leads to fatty liver disease encompassing steatosis, steatohepatitis and fibrosis. This places individuals at risk of developing cirrhosis, hepatocellular carcinoma or hepatic decompensation and also contributes to the emergence of insulin resistance and dyslipidemias affecting many other organs. Excessive accumulation of TG in adipose tissue contributes to insulin resistance as well as to the release of cytokines attracting leucocytes leading to a pro-inflammatory state. Pathological accumulation of cholesteryl ester （CE） in macrophages in the arterial wall is the progenitor of atherosclerotic plaques and heart disease. Overconsumption of dietary fat, cholesterol and carbohydrates explains why these diseases are on the increase yet offers few clues for how to prevent or treat individuals. Dietary regimes have proven futile and barfing surgery, no realistic alternatives are at hand as effective drugs are few and not without side effects. Overweight and obesity-related diseases are no longer restricted to the developed world and as such, constitute a global problem. Development of new drugs and treatment strategies are a priority yet requires as a first step, elucidation of the molecular pathophysiology underlying each associated disease state. The lipid droplet （LD）, an up to now over- looked intracellular organelle, appears at the heart of each pathophysiology linking key regulatory and metabolic processes as well as constituting the site of storage of both TGs and CEs. As the molecular machinery and mechanisms of LDs of each cell type are being elucidated, regulatory proteins used to control various cellular processes are emerging. Of these and the subject of this review, small GTPases belonging to the Rab protein family appear as important molecular switches used in the regulation of the intracellular trafficking and storage of lipids.展开更多
基金Supported by National Natural Science Foundation of China(31472260)
文摘With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.
文摘Abnormal intracellular accumulation or transport of lipids contributes greatly to the pathogenesis of human diseases. In the liver, excess accumulation of triacylglycerol （TG） leads to fatty liver disease encompassing steatosis, steatohepatitis and fibrosis. This places individuals at risk of developing cirrhosis, hepatocellular carcinoma or hepatic decompensation and also contributes to the emergence of insulin resistance and dyslipidemias affecting many other organs. Excessive accumulation of TG in adipose tissue contributes to insulin resistance as well as to the release of cytokines attracting leucocytes leading to a pro-inflammatory state. Pathological accumulation of cholesteryl ester （CE） in macrophages in the arterial wall is the progenitor of atherosclerotic plaques and heart disease. Overconsumption of dietary fat, cholesterol and carbohydrates explains why these diseases are on the increase yet offers few clues for how to prevent or treat individuals. Dietary regimes have proven futile and barfing surgery, no realistic alternatives are at hand as effective drugs are few and not without side effects. Overweight and obesity-related diseases are no longer restricted to the developed world and as such, constitute a global problem. Development of new drugs and treatment strategies are a priority yet requires as a first step, elucidation of the molecular pathophysiology underlying each associated disease state. The lipid droplet （LD）, an up to now over- looked intracellular organelle, appears at the heart of each pathophysiology linking key regulatory and metabolic processes as well as constituting the site of storage of both TGs and CEs. As the molecular machinery and mechanisms of LDs of each cell type are being elucidated, regulatory proteins used to control various cellular processes are emerging. Of these and the subject of this review, small GTPases belonging to the Rab protein family appear as important molecular switches used in the regulation of the intracellular trafficking and storage of lipids.
