The Study On The Function Of Transferrin Of Chinese Mitten Crab In The Pathogenesis Of Spiroplasma

The Chinese mitten crab,Eriocheir sinensis,is one of the most common freshwater crustaceans,which occupies a large proportion in Chinese freshwater aquaculture industry.With the development of intensive culture and improvement of intensification,the risk of its disease is also increasing seriously.These diseases are mainly caused by viruses,fungi and parasite.Among them,Spiroplasma eriocheiris as a novel type of pathogens can cause the crabs suffering from "Tremor disease",which results in decreased growth in aquaculture populations of E.sinensis and vast economic losses.The result of preliminary proteomic study on E.sinensis hemocytes infected Spiroplasma eriocheiris shown the expression of transferrin and ferrin have significantly changed.In addition,other reports have confirmed that transferrin and ferritin are involved in the immune response of hosts in other species.To sum up,we infer that Transferrin,Ferrinl and Ferrin2 may play an important role in the immune defense mechanism of the crabs.In this paper,gene cloning and bio informatics analysis were used to study the biological characteristics of EsTF.Subsequently,EsTF,EsFel and EsFe2 expression changes were detected after exogenous supplement iron in water and inject S.eriocheiris to explore the functions of these proteins in the process of the crab against the infection of the bacteria.In a word,these findings could serve as a theoretical basis for future studies on the susceptibility/resistance of E.sinensis to S.eriocheiris,as well as contribute to reduce the harm of S.eriocheiris.This study mainly includes the following two aspects:1.Full length cloning of EsTF protein and bioinformatics analysisIn this experiment,the whole length of EsTF was successfully amplified by the gene cloning and RACE technique.The results showed that the full-length cDNA of the EsTF gene was 2748 bp,including a 2193bp open reading frame which encodes 385 amino acids.The molecular weight is 79.59 kDa and isoelectric point is 6.87.The result of bioinformatics analysis shown EsTF contains a signal peptide at 1-19 amino acid site and two highly conserved TR_FER domains.Homologous sequence and evolutionary analysis show that EsTF has a close genetic relationship with other EsTF of invertebrates,which prove it is highly conservation in the evolution.In addition,we found that EsTF mRNA was highest transcripted in nerve tissue,also a high level in muscle and intestines.Combined with the above experimental results,we have a more comprehensive understanding of the biological characteristics of EsTF,laying a foundation for further research on immune function.2.Immune function study of EsTF?EsFel and EsFe2Transferrin and ferrtin,important iron metabolism regulation proteins,are responsible for the transport and storage of iron respectively.Numerous studies have shown that they can play important role in the innate immunity of invertebrates.The transcription levels of EsTF,EsFel and EsFe2 in the hemocytes were detected by qRT-PCR,and the transcription level of mRNAs were significantly higher than that in the control group.Similarly,the expression of EsTF,EsFel and EsFe2 were also increased after exogenous supplemental iron in water.After exogenous supplement iron and injection with S.eriocheiris,these three gene transcription of mRNA levels were still higher than that of PBS group,while lower than the S.eriocheiris control group and the iron control group.Besides,we also found that the copies number of S.eriocheiris in the experimental group was significantly reduced,and the death rate decreased,these results further demonstrate that EsTF,EsFel and EsFe2 may play a role in the immune defense mechanism of the crabs in response to S.eriocheiris.