The Cloning And Identify Of MLP And BBD Functional Gene Of Haemaphysalis Longicornis

Many pathogens such as bacteria, virus and protozotan can transmit by the hard ticks, which seriouslythreat the development of husbandry and the health of mankind. Currently, the most popular tick controlmethod is the application of acaricides. It is necessary to develop an alternative agent of tick control asthe growing number of disadvantages caused by acaricides. The study cloned and expressed the targetgene from Haemaphysalis longicornis, the most important hard tick in China, by the method of rapidamplification of the cDNA ends (RACE) and immunoscreening. To analyzed the biological function andassessed the potential for controlling ticks.The complete sequence of muscle LIM proteins (MLP) from Haemaphysalis longicornis, which wasscreened from a cDNA library constructed from partial-fed female ticks, was obtained by rapidamplification of the cDNA ends (RACE). The full-length of HLMLP was948bp. The HLMLP cDNAcontains321bp that encode106amino acids with a predicted theoretical isoelectric point (pI) of8.58and molecular weight of11.3ku. The nucleotide sequence of MLP and the deduced amino acid wereanalyzed by the bioinformatics software, and the complete open reading frames were amplified by PCRusing universal primer from ticks of Haemaphysalis qinghaiensis, Hyalomma anatolicum, Hyalommarufipes, Boophilus (Rhipicephalus) microplus, Demacentor silvarum respectively, the PCR product wasinserted into expression vector pGEX-4T-1to construct recombinant plasmid, and the recombinantplasmid was transformed into Escherichia coli BL21(DE3) for expression. The expression andreactogenicity of recombinant protein were analyzed by SDS-PAGE and Western-blot. The expressedfusion protein was approximately39.2ku in molecular weight. Western blot analysis showed that rabbitantiserum against the adults of H. longicornis could recognize the rHLMLP and the protein of differentticks. Real-time PCR analysis showed that this gene expressed in integument was significantly higherthan in other tested tissues of partially fed females, and the HLMLP gene expression is developmentallyregulated throughout the life cycle. Furthermore, the transcripts of the HLMLP mRNA weresignificantly downexpression in response to low temperature stimulation. The result of vaccination withrHLMLP and RNAi showed that there were no apparent obvious anti-tick effections.The full-length cDNA encoding gamma-butyrobetaine,2-oxoglutarate dioxygenase (HLBBD) wasobtained from H. longicornis by5′RACE and3′RACE. The full-length of HLBBD was1780bp. Thecomplete open reading frame (ORF) contains1353bp and coding for450amino acids with a predictedmolecular weight of45.0ku. The gene included functional domain of gamma-butyrobetaine,2-oxoglutarate dioxygenase. The predicted3D structure of HLBBD based on the structure of humangamma-butyrobetaine,2-oxoglutarate dioxygenase was constructed by Swiss-Model. Western Blotsanalysis showed that the expression product of HLBBD showed the strong reactogenicity. Real-timePCR analysis showed that the expressions of HLBBD were no obvious tissue specificity, and the genewas expressed in larval was lower than other different developmental stages. Additionally, thetranscripts of the gene were not significantly induced by blood feeding. RNAi experiment confirmed that the gene could significantly affect the engorgement weight and mortality in blood feeding, and theexceptional oviposition behavior was obeserved in treated groups. These results confirmed that thisprotein play essential role in the progress of tick feeding and production.