The Expression And Function Of Hf-FER1 And Hf-FER2 In Haemaphysalis Flava

Haemaphysalis flava,one of the common tick species in China,can attack many vertebrates,such as pigs,dogs,cattle and hedgehogs.It can harbor a variety of pathogens like Severe fever with thrombocytopenia syndrome bunyavirus,Tick borne encephalitis virus and Rickettsia,etc.,which poses a serious threat to animal husbandry production and public health.Ferritin is an iron binding protein,which has the function of storing iron ions and reducing the harm of iron.It is particularly important for ticks that live on blood-sucking.In this study,high-throughput sequencing was used to analyze the gene expression in the ovary of Haemaphysalis flava,and ferritin was selected as the target of the follow-up study;Unigenes of ferritin-1（Hf-FER1）and ferritin-2（Hf-FER2）were searched from the ovary transcriptome Library of Haemaphysalis flava,and their full length was cloned by rapid-amplification of c DNA ends（RACE）;The expression of Hf-fer1 and Hf-fer2 in different stages and different tissues of partially engorged and fully engorged females of Haemaphysalis flava were analyzed by real-time PCR;Recombinant Hf-FER1 and recombinant Hf-FER2 were prepared by prokaryotic expression system,and their characteristics were analyzed by non natural electrophoresis and iron chelation test;The Hf-fer1 and Hf-fer2 of fully engorged female H.flava were interfered with ds RNA to analyze their effects on the reproduction;The egg proteins of RNAi ticks were analyzed by LC-MS/MS technology to study the mechanism of decrease of incubation rate.The main results are as follows:1.Collect the ovaries of partially engorged and fully engorged females of Haemaphysalis flava and determine the expressed genes by Illumina method.138,711unigenes were obtained through sequence splicing,of which 72,043 unigenes（about 52%）were functionally annotated.In the process of blood-sucking,19,031 genes were up-regulated and 19,456 genes were down-regulated.Most of the differentially expressed genes were related to tick physiological activities such as ovarian development and egg maturation.The ovarian transcriptome data laid a foundation for the cloning of subsequent functional genes and the annotation of egg proteome.2.The full length of Hf-fer1 and Hf-fer2 were cloned by RACE;The results of real-time PCR showed that Hf-fer1 and Hf-fer2 genes were expressed in all stages of Haemaphysalis flava.With the blood-sucking of female ticks,the expression levels of Hf-fer1 and Hf-fer2were significantly up-regulated（P<0.05）,especially the relative expression of Hf-fer2 in partially engorged and fully engorged female ticks was 1,000-2,000 times higher than that in non sucking female ticks;The ORF of Hf-fer1 and Hf-fer2 was inserted into p ET-32a（+）,respectively,and r Hf-FER1 and r Hf-FER2 were successfully expressed.The results of nature electrophoresis showed that r Hf-FER1 and Hf-FER2 could self assemble to form 24 polymer;The iron chelation test showed that 1 g of r Hf-FER1 or r Hf-FER2 could bind divalent iron ions about 3.12 x 10^-4 mol.The results indicated that Hf-FER1 and Hf-FER2 play an important role in the iron storage of Haemaphysalis flava,and they are important substances to maintain iron homeostasis in tick life cycle.3.The double stranded RNA of Hf-fer1 and Hf-fer2 was injected into fully engorged females of Haemaphysalis flava through the valve plate for gene interference,respectively,and the expression of the two genes was determined by real-time PCR.The results showed that both genes were effectively silenced;The gene silencing of Hf-fer1 or Hf-fer2 caused that the oviposition of Haemaphysalis flava decreased,some of the eggs were deformed,and the the eggs incubation rates decreased by 42.73%and 72.66%,respectively.4.The egg protein components of RNAi ticks were analyzed by LC-MS/MS technology.The results showed that the interference of Hf-fer1 caused the up regulation of carboxypeptidase,TIL domain-containing protein and serine protease inhibitor,while the down regulation of superoxide dismutase in the tick eggs;Hf-fer2 interference caused up regulation of carboxypeptidase and cathepsin,absence of Hf-FER2,and down regulation of superoxide dismutase and serine protease inhibitors in the tick eggs.These results indicated that the silencing of Hf-fer1 or Hf-fer2 would lead to the disorder of tick egg protein components,resulting in the imbalance between protease and protease inhibitor,and the imbalance between oxidation and reduction.To sum up,this study researched that the role and function of Hf-FER1 and Hf-FER2 in the blood sucking and reproductive oviposition of ticks,and found that the gene expression of Hf-fer1 and Hf-fer2 was up-regulated with the blood sucking of female ticks,and r Hf-FER1 and r Hf-FER2 could chelate a large number of divalent iron ions,indicating that they are important substances to maintain iron homeostasis in tick life cycle.The interference of Hf-fer1 or Hf-fer2 resulted in the decrease of egg laying and egg hatching rate of fully engorged female ticks.The analysis of proteomics analysis of tick eggs showed that the silencing of Hf-fer1 or Hf-fer2 would lead to the disorder of tick egg protein components,resulting in the imbalance of protease and protease inhibitor,as well as the imbalance of oxidation and reduction,and affecting the incubation of tick eggs.This study will be beneficial to the exploitation and application of Hf-FER1 and Hf-FER2 in anti-ticks vaccines and tick borne disease control.