| Roughskin sculpin, Trachidermus fasciatus Heckel(Scorpaeniformes:Cottidae), is a carnivorous fish with seawater-freshwater migratory habits. As one of the second order of protected aquatic animals in China, roughskin sculpin is of great biological, ecological, and economic importance. Considering its serious decline during the last few decades, aquaculture has become an important strategy for recovering its population in the wild. However, roughskin sculpin possesses unique pathologic feature due to its unique biological and physiological characteristics. Up to now, few studies have reported on the molecular mechanisms of stimuli response of roughskin sculpin.In addition, aquaculture animal welfare is becoming a hot issue all over the world. Huntingford said that the animal welfare is to ensure animal live without painful elements. How to avoid the stress reaction is very important, because it waste energy. The sum of nonspecific response of the body on the outside or inside of various stimuli are called stress reaction, it has an important impact on aquaculture life, too strong or too long reaction will make an adverse effect on breeding, so it’s bad for animal welfare. So in order to raise the breeding efficiency, avoiding causes of stress reaction is very important.HSPs are a family of highly conserved proteins that play a key role in protective reaction. They are highly conserved during evolution and of utmost importance functionally. HSPs perform essential biological functions under both normal and stress conditions. In normal conditions, heat shock proteins primarily act as molecular chaperones, involved in protein metabolism, cell cycle regulation, and apoptosis and so on. When organisms are stressed by high temperature, crowding, hypoxia, heavy metals, hunger, trauma, viral and bacteria infection, HSP expression levels would significantly increase, enabling organisms to resist the insults of adverse stressors so as to maintain the homeostasis and survival of cells.So monitoring the level of stress responses is really a significative problem that all the aquaculture experts must face, and finding a physiological or biochemical index is especially in urge need. If we can choose a molecular index which is easy to obtain, and the method to determine the correlation between the expression level and the degree of stress reaction is precise, we can directly put it into practice.HSPs have been observed in all organisms, including fish. HSPs could be induced in cells when exposed to stress, and are considered to be involved in a general mechanism that helps cells to survive under many lethal stresses. Extensive studies on model species have discovered several HSP families, which are named based on the molecular mass (kDa). Three major families of HSPs are HSP90(85-90kDa), HSP70(68-73kDa) and low-molecular-mass HSPs (16-24kDa). HSP70is known to help fold nascent polypeptide chains, act as a molecular chaperone and mediate the repair and degradation of altered or denatured proteins and so on. There have been several efforts to validate the use of the HSP response as an indicator of stressed states in fish now.As a consequence of their abundance and the high level of homology among various organisms, scientists have discovered that HSPs involve in immune response. Research showed that many diseases relate to the expression of HSPs. HSPs induced in host could increase resistance to the environmental stress. HSPs are major targets of host immune response. This is particularly true for the members of HSP70family. The70-kD heat-shock protein (HSP70) is the most abundant HSP and its structure is remarkably conserved among various organisms. HSP70could be induced in cells when exposed to stress and it is surprised that HSP70could be used as an indicator of stress states in fish, such as infection.At the same time, with the obtained influence under various environmental stresses, the induction of HSP70synthesis can make biological, salty and heat resistance. So the organisms can adapt to the environment. According to HSP70gene sequence (AY120894), we design and synthesize a pair of primers, then with the total RNA of liver tissues in roughskin sculpin as template, we get a partial sequence of roughskin sculpin HSP70cDNA, the length of which is480bp. And its GeneBank accession number is KC342053. According to the known sequence, the cloning total sequence had been made by the RACE method. The results of sequence analysis indicated that the sequence with red snapper(Lutjanus sanguineus), a highly homologous to Lutjanus sanguineus and so on. To obtain the roughskin sculpin cDNA full-length2138bp, open reading frame of1947bp, encoding649amino acids. Amino acid alignment shows, partial sequence of roughskin sculpin with other known homologue of82%-99%, and amphibians, birds and mammals for75%-86%homology.In order to study whether HSP70can be used as a molecular marker for stress monitoring, with the obtained gene partial sequences of HSP70roughskin sculpin, and a sequence of roughskin sculpin of P-actin gene (HM449124) as a reference, using fluorescence quantitative PCR technology, detection of differential expression of roughskin sculpin muscle, intestine, brain, skin, liver, gonad, heart, gills, fins9organizations. The results show that HSP70is widely expressed in roughskin sculpin in9tissues, including the highest expression level in the fins, expression in the gonads, gill, brain, muscle, liver, skin, intestine decreasing, in the heart of the lowest expression. Expression of HSP70in fin could be index of roughskin sculpin stress level. In addition, HSP70of8organs or tissues changes during four seasons also have been detected, it is found that HSP70of8tissues in summer is the highest, and HSP70of3tissues in spring is highest, HSP70in all tissues in autumn is the lowest, which provide scientific data for the breeding of roughskin sculpin, and gets the new insight for the fish environment index control. For further study, whether HSP70can make a good use in theory or in practical breeding needs further study. |
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