| Grass carp(Ctenopharyngodon idella) is one of the most important freshwater aquaculture species in China. Because this aquaculture fish is susceptible to a series of stresses throughout the breeding process, high mortality of fish and enormous economic loss often happen. Glucose regulated protein (GRP) is a member of heat shock protein (heat shock protein, HSP) subfamily. A protective mechanism used for cells to adapt to stress of endoplasmic reticulum (ER) is the induction of members of the glucose-regulated protein family. GRP is a kind of molecular chaperone in endoplasmic reticulum which has very important effect on protein folding, assembly and trafficking. GRP78plays a vital role in antioxidant defense system, including hydrogen peroxide enzyme, peroxidase resistance and the toxic effects of heavy metal, can be used for diagnosis of heavy metal pollution index. In order to understand the molecular mechanism response to stress, we carried out the research of the grass carp GRP78and oxidase on thermal stress and heavy metal lead stress.1、Quantitative RT-PCR analysis showed that the expression of CiGRP78was ubiquitous in all detected tissues and up-regulated after heat shock at34℃or cold stress at4℃. It mainly displayed greater level in the liver, kidney tissues. After treated by lead nitrate at0.25mmol·L-1for24h, the expression level of GRP78was increased in all tissue. Compared to other tissues, the expression in liver tissue increases most obvious.2、In order to understand the function of CiGRP78responded to thermal stress and heavy metal lead stress, we made the cell protection experiment of heat shock at34℃or cold stress at4℃. The results showed, CiGRP78not only can contribute to protect prokaryotic cells against both thermal and cold extremes, but also play a protective role in mouse myeloma cells and grass carp kidney cells. At28℃for36h, the survival rate of CIK cell transfect or un-transfect with GRP78had no significant change, this illustrates of transfect with vectors or target gene do not affect the growth of CIK cell. In low concentrations of lead for36h, the survival rate of three kinds of CIK cells is significantly different. The survival rate of transfect with target gene in cell is always higher than un-transfect and transfect with vector in cell. It is still higher after36h. In addition, GRP78showed a protective effect on cell suffer from heavy metal stress. Although the mechanism is not clear, upon the high concentrations of Pb2+stress in24h gene transfection, cell survival rate was significantly higher than that of other two kinds of cells, but there were no significant difference at36h.3、Lead and its compounds are common heavy metal pollutions in water which are harmful to fish. In order to study the influence of lead and its compounds on the resistance ability of blood serums oxidation from grass carp, we used Pb2+solution of different concentrations to treat grass carp. The change of hydrogen peroxide (H2O2) content and the activities of catalase (CAT), myeloperoxidase (MPO) in the grass carp blood serum had been investigated. The results indicated that Pb2+in the lower concentration would induce the rise of H2O2in grass carp serum. With the concentration of Pb2+increased, the H2O2content was at a higher level. When the concentration of Pb2+was in0.25mmol·L-1, H2O2content was the maximum. The activities of CAT and MPO increased weakly induced by Pb2+in0.01mmol·L-1concentration, and then they both reduced gradually as the increase of concentration of Pb2+. Thereinto, in the0.01mmol·L-1group, the activity of CAT reached the peak at48h, then it was reduced. The activity of MPO was enhanced generally and reached the peak at72h and maintained at a high level at96h. The activities of CAT and MPO were inhibited by high concentration of0.25mmol·L-1Pb2+treated. The inhibition of CAT was the highest at48h then it recovered to normal level at96h. The activity of MPO had not recovered. The lowest activity presented at72h. |
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