Full-length Cloning And Vector Expression Analysis Of Atg4 And Atg5 In H.flava Tick

Ticks are parasites that feed on blood and have direct and indirect harm to humans and animals.Autophagy is a physiological process that is necessary for normal cells and regulated by autophagy-related genes.Nowadays,many scholars have analyzed and studied some autophagy-related genes and their expression levels of H.longicornis,R.microplus,I.scapularis,and R.haemaphysaloides.and found that autophagy can be affected in starvation and embryonic state.Play a role in different processes such as development,growth and nutrient utilization.In this study,H.flava as the research object,the full-length sequences of HfATG4 and HfATG5 were cloned by the RACE method,and the expression levels of HfATG4 and HfATG5 in different growth stages,different satiation states,and different tissues and organs were determined by RT-PCR method.Provide a basis for the development of a broad-spectrum anti-tick vaccine.The main research contents and methods are as follows:1.Based on the results of proteomic analysis,9 ATG protein sequences(Atg3,Atg4,Atg5,Atg6,Atg7,Atg8,Atg13,Atg16,Atg101)were found from the ovary transcriptome library of H.flava(PRJNA600997).The results showed that the different ATG protein sequences had great differences.But sequences of the same ATG from different ticks are highly similar.2.The 5’ and 3’ ends of Cluster-24081.57394 and Cluster-24081.6376 were amplified by RACE method,and the full-length c DNAs of HfATG4 and HfATG5 were obtained.HfATG4 is 1361 bp in length,open reading frame(ORF)is 1164 bp,encoding protein 387aa;HfATG5 is 1813 bp in length,ORF is 813 bp,encoding protein 270 aa.3.The expression levels of HfATG4 and HfATG5 measured by RT-PCR at Larvae,Nymphs and adult stages were as follows: Larvae > Femina > Male > Nymphs.Expression levels in four different engorged states: The expression of HfATG4 was the highest in 1/4engorged female ticks;The expression of HfATG5 was the highest in 4/4 engorged female ticks.The expression of HfATG4 in salivary gland,ovary,midgut and body shell was as follows: the expression of fully engorged was higher than that of semi-saturation;The expression of HfATG5 in salivary gland,ovary,midgut and was as follows: the expression of fully engorged was higher than that of semi-saturation,The expression of the body shell was higher in half-satiated blood than in satiated blood.HfATG4 was up-regulated and HfATG5 was down-regulated in the early stage of embryonic development,and was basically stable in the late stage.The results of the study show that the types of autophagy proteins in H.flava are abundant,which can provide a material basis for screening candidate molecules for the prevention and control of tick-borne diseases.The differential expression of HfATG4 and HfATG5 genes in different tissues and organs in different growth stages and different states of full blood.It is suggested that it may be involved in the growth and development of ticks and the process of blood-sucking digestion.It has potential as a candidate molecule of anti-tick vaccine.