Gene Cloning, Prokaryotic Expression And Funcational Analysis Of Ferritin From The Hyalomma Rufipes And Haemaphysalis Doenitzi

Ticks are blood-sucking ectoparasites with a very wide range of hosts.In this study,Hyalomma rufipes is a two-host tick,which is commonly found in desert and semi-desert areas and generally distributed in Inner Mongolia,Shanxi,Ningxia and Xinjiang.Haemaphysalis doenitzi is a tri-host tick,common in the wild and jungle,mainly distributed in Hainan,Fujian,Taiwan,Yunnan and other places in China.Ticks can transmit a variety of pathogens in the process of sucking blood,which poses a great threat to human health and the development of animal husbandry.At present,the prevention and control of tick-borne diseases is mainly based on chemical control,but long-term use of chemical drugs will cause environmental pollution,and the drug resistance of tick with long-term adaptation is also a difficult problem to solve in chemical control,so biological control,immune control and genomics have gradually become the focus of tick control research.Ferritin is widely found in animals,plants and microorganisms.It is an important protein for organisms to maintain iron homeostasis and plays an indispensable role in detoxification,resistance to oxidative stress and innate immune response.Ticks need to against a large amount of iron and exogenous microorganisms from the host blood when feeding.The mechanism of tick's ferritin in blood meal digestion and immune defense is a meaningful study.In this study,three kinds of ferritin from Hyalomma rufipes and Haemaphysalis doenitzi were systematically studied by using gene cloning,prokaryotic expression,quantitative Real-time PCR,RNAi and other biotechnology.Three ferritin genes were cloned which named HrFer1,HrFer2 and HdFer,and the number of nucleotides in the open reading frame is 519 bp,573bp and 525 bp,respectively.They could code 170,190 and 172 amino acid,respectively.And the theoretical pI of their proteins was 4.94,5.51 and 4.71,respectively.The predicted results of signal peptide showed that HrFer1 and HdFer do not contain signal peptide,but the amino acid sequence of HrFer2 has a signal peptide in 1-15 aa.The amino acid sequences of HrFer1,HrFer2 and HdFer were compared with ferritin of other animal,showed a close evolutionary relationship with ferritin H subunit,and the evolutionary relationship with ferritin M subunit and L subunit are verydistant.SDS-PAGE showed that the molecular weight of the three recombine proteins were about 40 kDa,41 kDa and 40 kDa,respectively,which were consistent with the predicted sizes.With the expression conditions were further optimized,the recombinant proteins rHrFer1,rHrFer2 and rHdFer were successfully purified by 250 mM,200 mM and 250 mM imidazole,respectively.The rHrFer1,rHrFer2 and rHdFer all showed concentration-dependent activity while analysis the iron chelating activity and antioxidant activity of the recombinant proteins.The results of Quantitative Real-time PCR showed that the three ferritin genes had high relative expression levels in the ovary and midgut of ticks,which were significantly different from other tissues.RNA interference technology was used to explore the effect of ferritin on biological traits of ticks.It was found that interference with HrFer1 and HrFer2 genes had significant effects on the engorgement weight,spawning volume and mortality of ticks.In addition,interference with the HrFer2 gene can significantly affect the feeding time of ticks.Compared with HrFer2,HrFer1 have a more significant effect on the spawning volume.In order to explore the role of ferritin's high expression in the midgut,this study analyzed the midgut microbial communitys of ticks after RNA interference.The results showed that after interfering with the ferritin gene,the microbial communitys of the midgut were changed,and the relative abundance of some symbiotic bacteria tended to decrease,while that of some pathogenic bacteria tended to increase.These results suggest that ferritin may play a role in maintaining the homeostasis of microbial community structure in midgut community.The low temperature stress treatment was carried out for Hyalomma rufipes,it was found that the relative expression of ferritin decreased significantly in the low temperature environment,indicating that HrFer1 and HrFer2 have certain regulatory effects on the resistance to low temperature stress.The above results provide a reference for comprehensively revealing the mechanism of ferritin in ticks and lay a foundation for exploring its specific functions.