采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b(+)中,然后转化大肠杆菌BL21(DE3),用1mmol/L IPTG在不同温度及时间下进行诱导表达。采用Ni-NTA His Bind Resins亲和层析和DEAE-S...采用PCR方法扩增团头鲂组成型HSC70和诱导型HSP70基因完整的编码区片段,并分别克隆到原核表达载体pET-22b(+)中,然后转化大肠杆菌BL21(DE3),用1mmol/L IPTG在不同温度及时间下进行诱导表达。采用Ni-NTA His Bind Resins亲和层析和DEAE-Sepharose FF阴离子交换柱层析对目的蛋白进行纯化,并进行SDS-PAGE和Western-blotting分析。结果表明,成功构建了团头鲂两种重组表达质粒pET-22b(+)/Ma-HSC70和pET-22b(+)/Ma-HSP70,表达融合蛋白的相对分子量均约为72kDa,并能与兔抗人HSP70多抗进行特异性结合,这两种HSP70s融合蛋白经纯化后的纯度均达到95%以上。本实验选择融合蛋白Ma-HSC70在25℃和Ma-HSP70在30℃下分别诱导7h作为可溶性表达的最佳条件。展开更多
Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned f...Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.展开更多
Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characte...Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P <0.05) at 16 h,and were then upregulated to near the initial level at 10 d.展开更多
In order to find out the change pattern of phytoplankton community structure in pond industrial eco-aquaculture system and explain its ecological mechanism,tests were carried out using Megalobrama amblycephala as aqua...In order to find out the change pattern of phytoplankton community structure in pond industrial eco-aquaculture system and explain its ecological mechanism,tests were carried out using Megalobrama amblycephala as aquaculture species with two stocking densities.Shannon-Wiener diversity index and Pielou uniformity index were used to study the phytoplankton community structure in aquaculture area( including low stocking density aquaculture area and high stocking density aquaculture area),inlet area,outlet area,purification area,recirculation area,back area of the pond industrial eco-aquaculture system.The results showed that a total of 92 species of 5 phyla were identified,including 46 species of Chlorophyta,15 species of Cyanophyta,15 species of Euglenophyta,10 species of Bacillariophyta,5 species of Cryptophyta,and 1 species of Pyrrophyta.The numbers of phytoplankton species,biodiversity indexes,uniformity indexes,Chlorophyta biomass and its proportion in total phytoplankton followed the order of inlet area,purification area,recirculation area,back area > low stocking area > high stocking area > outlet area.The total biomass of phytoplankton,Cyanophyta biomass and its proportion in total phytoplankton followed the order of inlet area,purification area,recirculation area,back area < low stocking area < high stocking area < outlet area.However,all these parameters showed no significant differences or change rules among inlet area,purification area,recirculation area,back area.It could be concluded according to the biodiversity that the water quality in outlet area was at middle pollution level,the water quality in high stocking area was in light pollution level,and the water quality in other 5 areas were at clean level,which suggested that the water quality was in good states except that in outlet area,and the pond industrial eco-aquaculture system functioned well.展开更多
基金supported by grants from the Key Technologies Research and Development Program of China(2012BAD26B02)National Science Foundation of China(31272633+2 种基金31201760)Special Funding from the Ministry of Agriculture of China(201203081,201203086)Shanghai University Knowledge Service Platform(ZF1206)
文摘Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.
基金Supported by the National Natural Science Foundation of China(No.31302222)the Earmarked Fund for China Agriculture Research System(No.CARS-46)+1 种基金the Freshwater Fisheries Research Center,CAFS Grant(Nos.2013JBFM10,2013JBFM03)the Natural Sciences Foundation of Jiangsu Province(No.BK2011182)
文摘Mucins are important components of mucus,which form a natural,physical,biochemical and semipermeable mucosal layer on the epidermis of fish gills,skin,and the gastrointestinal tract. As the first step towards characterizing the function of Muc2,we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp,and analyzed its tissue-specific expression pattern by quantitative real-time PCR(qPCR). The obtained sequence comprised 41 bp 5′-untranslated region(5′-UTR),2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains(VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes(β- Actin,RPI13α,RPII,18S) for the qPCR,R PII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine,in the order(highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs(brain,liver,kidney,spleen,skin and gill). Furthermore,after 20-days of starvation,M. amblycephala Muc2 expressions after refeeding for 0h,3 h,16 h,3 d,and 10 d were significantly decreased in the three intestinal segments(P <0.05) at 16 h,and were then upregulated to near the initial level at 10 d.
基金Supported by the Fund for Independent Innovation of Agricultural Sciences in Jiangsu Province(CX(16)1004)the National Key Technology R&D Program of China(2015BAD13B03)
文摘In order to find out the change pattern of phytoplankton community structure in pond industrial eco-aquaculture system and explain its ecological mechanism,tests were carried out using Megalobrama amblycephala as aquaculture species with two stocking densities.Shannon-Wiener diversity index and Pielou uniformity index were used to study the phytoplankton community structure in aquaculture area( including low stocking density aquaculture area and high stocking density aquaculture area),inlet area,outlet area,purification area,recirculation area,back area of the pond industrial eco-aquaculture system.The results showed that a total of 92 species of 5 phyla were identified,including 46 species of Chlorophyta,15 species of Cyanophyta,15 species of Euglenophyta,10 species of Bacillariophyta,5 species of Cryptophyta,and 1 species of Pyrrophyta.The numbers of phytoplankton species,biodiversity indexes,uniformity indexes,Chlorophyta biomass and its proportion in total phytoplankton followed the order of inlet area,purification area,recirculation area,back area > low stocking area > high stocking area > outlet area.The total biomass of phytoplankton,Cyanophyta biomass and its proportion in total phytoplankton followed the order of inlet area,purification area,recirculation area,back area < low stocking area < high stocking area < outlet area.However,all these parameters showed no significant differences or change rules among inlet area,purification area,recirculation area,back area.It could be concluded according to the biodiversity that the water quality in outlet area was at middle pollution level,the water quality in high stocking area was in light pollution level,and the water quality in other 5 areas were at clean level,which suggested that the water quality was in good states except that in outlet area,and the pond industrial eco-aquaculture system functioned well.
