| As an important fishery resource,temperature is one of the important factors affecting the growth and development of Fenneropenaeus chinensis,Its culture season and region are limited by low temperature.Therefore,in order to improve the low temperature tolerance of Fenneropenaeus chinensis,it is necessary to carry out in-depth research.The purpose of this study is to provide theoretical basis for molecular breeding of new low temperature tolerant varieties of Fenneropenaeus chinensis through exogenous expression of low temperature tolerance gene and verification of freezing resistance of Fenneropenaeus chinensis.Based on Wang Jing et al’s previous transcriptome sequencing analysis of Fenneropenaeus chinensis under low temperature stress,the up-regulated candidate genes were sampled under gradient low temperature stress in 253N families with excellent freeze resistance,and CL161.Contigl,Unigene889,Unigene26823,CL2560.Contigl and CL2861.Contig5 5 genes were verified by Real time RT-PCR.The results showed that the expression of the five genes in the low temperature stress group was higher than that in the normal temperature control group,and the lower the temperature was,the higher the expression was.The prokaryotic recombinant expression vectors of pET28a-161,pET28a-889 and pET28a-26823 were successfully constructed.The soluble expression was induced at 16 ℃ and the final concentration of IPTG was 0.2 mM for 16 h,and the inclusion body expression was induced for 12 h at 25℃ and 0.6 mM at the final concentration of IPTG.The soluble expression of pET28a-26823 was induced for 16 h at 16℃ and the final concentration of IPTG was 0.2 mM,and the inclusion body expression was induced at 37℃ and 1.0 mM for 12 h.The expression of inclusion body was induced by pET28a-889 at 16℃ and the final concentration of IPTG was 0.2 mM for 16 h.The recombinant strain pET28a-161,pET28a-889,pET28a26823 was verified by Real timeRT-PCR after low temperature treatment,and the target gene expression of the three recombinant strains was up-regulated at low temperature.In the experiment of freezing resistance,the recombinant strains of pET28a-161 and pET28a-26823 were treated with low temperature(0℃,-20℃)for 24 hours and then resuscitated.The cell growth rate and final concentration of the recombinant strains were higher than those of the control strains.The recombinant strains of pET28a-161,pET28a-26823 and the control group were induced and cultured at-20℃.The results showed that the colony of the recombinant strain was significantly more than that of the control group with the increase of time.The recombinant pET28a-161 inclusion body renatured proteins of different concentrations were added to the Escherichia coli solution incubated at 0℃.The results showed that the survival rate of Escherichia coli with renatured protein was significantly higher than that without renatured protein,and there was no significant difference in the amount of renatured protein at 10 μg/mL-50 μg/mL.The results showed that the recombinant protein could exert strong antifreeze effect at low concentration(10 μg/mL).The eukaryotic recombinant expression vector pcDNA3.1(+)-889 was successfully constructed by homologous recombination with human liver cancer cell line Huh7 as the expression host.The recombinant cells with an initial concentration of 105/mL and the control group were exposed to 0℃ low temperature stress for 12 h,24 h,36 h and 48 h,and then 10 μL CCK8 reagent was added to each well for different time(0.5 h,1 h,4 h).The results showed that the cell viability of the experimental group was stronger than that of the control group,and the difference was the most obvious after 24 h of low temperature stress.The cell viability of the experimental group was still slightly higher than that of the control group at 12 h,36 h and 48 h of low temperature stress,but the difference was not significant.The subsequent resuscitation activity of the recombinant cells and the control group were measured after resuscitation at 37℃ for 12 h,24 h 36 h and 48 h,respectively.The cell viability of the experimental group was higher than that of the control group at all resuscitation times.The difference between the two groups became more obvious with the prolongation of resuscitation time,indicating that the target gene has a significant antifreeze protective effect.The E.coli solution with different concentrations of recombinant cell lysate(100 μg/mL,200 μg/mL,300μg/mL)was treated at-20℃.The results showed that the number of colonies in the experimental group was higher than that in the control group,and the number of colonies increased with the increase of protein concentration.The results showed that Unigene889 played a role in freezing resistance in Huh7 cells,and in a certain range,the freeze resistance effect of the recombinant protein was positively correlated with its concentration. |
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