| In this paper,Cher ax quadricarinatus were used as the research object,and biotechnology such as biochemical,immunotoxicology and molecular biology were used as the research method,by controlling the external temperature conditions,the effects of temperature change especially low temperature on the growth and immunization of Cherax quadricarinatus were studied.The results are as follows:1.Effects of different temperatures on growth performance and antioxidant indexes of Cherax quadricarinatusIn order to explore the effects of temperature on the growth performance,antioxidant index,immunity of the Cherax quadricarinatus,the experiments were carried out for 8 weeks.Four temperature groups were set at 10℃.16℃,22℃,28℃,3 replicates per group.The results showed that the survival rate and growth rate of the Cherax quadricarinatus were significantly different at different temperatures.At low temperature(10℃),the feeding activity,growth and survival of the Cherax quadricarinatus were significantly inhibited.While the suitable temperature,can significantly promote the survival and growth of Cherax quadricarinatus.The effects of temperature on the activities of digestive enzymes and antioxidant enzymes in hepatopancreas,gills and stomach tissues of Cherax quadricarinatus were also studied.The results showed that the activities of amylase and trypsin were the highest at 28℃,The activities of amylase and trypsin were the lowest in the 10℃group,which was significantly lower than that in the other groups.SOD activities of the three tissues reached the peak at 28℃,and the activities of SOD were the lowest at 10℃.The results showed that low temperature could significantly inhibit the activity of digestive enzymes and antioxidant enzymes of Cherax quadricarinatus which affected their growth and survival.2.Effects of different temperature on immune Indexes of Cherax quadricarinatusThe ACP,AKP and LSZ activities in hepatopancreas,gills and muscle tissues of Cherax quadricarinatus were studied.The ACP and AKP activities were the highest at 22℃and the ACP and AKP activities at 10℃ were significantly lower than those in the other groups(P<0.05).The activity of lysozyme was the lowest at 10 ℃,and the activities of lysozyme at 22℃ were not significantly different from that at 28 ℃(P>0.05).The results showed that low temperature could significantly inhibit the immune-enzymatic activity of Cherax quadricarinatus,thus causing damage to its immune system.3.Effects of different temperature on disease resistance of Cherax quadricarinatusBy injection of 0.1ml and 1.0 ×108 cfu/ml of A.hydrophila,the activities of SOD were significantly different in different temperature groups.The results showed that at 10℃ A.hydrophila infection ability was very weak,so SOD enzyme activities were relatively strong,but the activities of SOD were decreased with the prolongation of infection time,which indicated that toxin protein of A.hydrophila were not expressed at low temperature,the infection capacity was relatively weak,but if long time in low temperature conditions,will also significantly affect the activity of immune enzymes.The activities of SOD at 22℃ and 28℃ were gradually increased to the original level with the prolongation of infection time,which indicated that the suitable temperature could significantly enhance the disease resistance of Cherax quadricarinatus.4.α-amylase gene cloning and expression analysisBy molecular biology technique,A fragment of the a-amylase gene of the Cherax quadricarinatus was cloned,which was A fragment of 970bp,open reading frame of 528bp,and encoded 175 amino acid residues.The results showed that the homology of the sequences was 89%and 85%similarity compared with that of Procambarus clarkii and Litopenaeus vannamei,analysis of its biological origin was found to be closely related to Procambarus clarki.The results of RT-PCR showed that the expression levels of a-amylase gene were significantly different in different temperature groups,at 28℃ the mRNA expression levels were the highest and at 10℃ were the lowest,which was significantly lower than that in the other groups(P<0.05),indicating that the a-amylase gene was involved in the growth process of Cherax quadricarinatus,which had a significant effect on the growth of Cherax quadricarinatus.5.HSP70 and Mn-SOD expression analysisThe results of RT-PCR showed that different temperature had a significant effect on the expression of two genes.HSP70 was highly expressed at 10℃ and 28℃,Mn-SOD was the lowest at 10℃.The results showed that the expression of HSP70 was induced by high temperature and low temperature environment.The Mn-SOD was induced at the appropriate temperature. |
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