Effect Of Low Temperature Stress On Physiological And Biochemical Indexes And Hsp70 Gene Expression Pattern Of Nibea Albiflora

To investigate behavioral, histological, physiological and biochemical indexes and the expression of the related cold resistant genes response of N. albiflora under the low temperature stress. We confirmed the semi-lethal low temperature of N. albiflora through the methods of experimental ecology. This research studied the impact of behavior, biochemical indexes and the related physiological indexes of N. albiflora. At the same time, We Cloned and separated Hsp70 gene core sequence of N. albiflora and studied its tissue distribution and before the low temperature stress, the differential expression of genes and proteins of heat shock protein70(Hsp70). The results are as follows:(1) Four treatments were set up, and 18℃ was as control and 5.5℃, 6.5℃, 7.5℃ and 8.5℃ were experimental groups. We studied the semi-lethal temperature in 12 hours and the changes of histology and behavior of N. albiflora under different temperature. The results showed that: under the condition of 5.5℃ and 6.5℃ temperature, experimental fish began to appear unbalance within a short time, floating in a mess and fully died within 6h and 9h respectively; under the condition of 7.5℃ temperature, experimental fish due to stress response appeared to be unbalance and roll over in 8 min. After 17 min, the experimental fish began to recover, while treated for 12 hours, half of the fish died. But the fishes of treatment 8.5℃ and 18℃ were normal. So the semi-lethal temperature was confirmed to be 7.5℃. Selected muscles and gills from fish in the control group(18℃) and low temperature treatment group(7.5℃) fish for paraffin sections which showed that low temperature stress can lead to swelling of the gill, blood vessel blockage, water deterioration and bubble deterioration of gill lamella, a large number of epidermal cells damaged and so on. The muscle fibers become swelling, and muscle fiber gap become widening and loose and so on. So, the results indicate that extreme low temperature can cause serious hurt to yellow croaker organization.(2) In order to investigate the effects of low temperature treatment on the physiological function of juvenile N. albiflora, we investigated the effect of cold stress on activities of antioxidant enzymes and Na~+/-K~+-ATP enzyme in juvenile N. albiflora. Four treatments were set up, and 18 ℃ was as control and 8 ℃, 10 ℃ and 14 ℃ were experimental groups. We measured the activities of antioxidant enzymes, Na+ K+-ATPase enzymes and Hsp70 content of N. albiflora at different temperatures. The results showed that the temperature significantly influenced the SOD and CAT enzyme activities, and Na~+/-K~+-ATP enzyme activities and Hsp70 content of N. albiflora. The results showed that antioxidant enzyme activities increasedin the beginning and then decreased under the low temperature stress, and at 72 h post-stress, antioxidant enzyme activities in low temperature treatments were at the same level as the control(P>0.05). Na~+/-K~+-ATP enzyme activities in the muscles of low temperature treatments showed the trend of decreasing firstly and then rising during the process of cold temperature stress. The Na+/-K+-ATP enzyme activities of 10℃ and 14℃ treatments could recover and have no significant differences at 72 h post-stress compared with the 18℃ treatment, but the activities of Na~+/-K~+-ATP enzyme in 8 ℃ treatment remained lower at 72 h post-stress. Therefore, SOD, CAT and Na~+/-K~+-ATPase are involved in the process when N. albiflora responding to low temperature stress, and they could be the markers corresponding to cold stress in N. albiflora.(3) RT-PCR and primers were used to clone the core nucleotide sequence of cDNA of Hsp70 gene. The sequence length of Hsp70 was 2143 bp, which contained an open reading frame(ORF) of 1917 encoding 639 amino acid(aa) residues, a 5’-untranslated region(UTR) of 128 bp, and a 3’-UTR of 98 bp. The nucleic acid homology analysis showed 99 % similarity between the Hsp70 of Nibea and large yellow croaker, drumfish. Semi-quantitative RT-PCR method was used to detect expression differences of Hsp70 in liver, gills, spleen and muscle tissue. The results showed that Hsp70 mRNA was expressed in all organizations, but varied in expression. Expression in gill and muscle was highest, followed by the spleen, liver least. The effect of expression of Hsp70 mRNA at low temperature(10℃) stress was studied through real-time PCR method.The results showed that the levels of Hsp70 mRNA of muscle under low temperature stress levels increased firstly and then decreased, suggesting expression of this gene was regulated by low temperature stress, and the sequence cloned in this study is core sequence of inducible Hsp70 c DNA. The study on protein expression differences of Hsp70 under low temperature stress in muscle suggested that the Hsp70 contents in different treatments were also impacted by the low temperature, and show the trend of increasing firstly and then decreasing during the process of cold temperature stress. Compared with the control, the Hsp70 contents of 10 ℃ and 14 ℃ treatments recovered and showed insignificant differences at 72 h post-stress. However, the 8℃ treatment Hsp70 contents remained higher than that of control at 72 h post-stress.