Differential Expression Analysis of RLCht1 Gene in Rhipicephalus linnaei and Detection of Enzyme Activity
[Objective]The molting process of ticks was closely related to chitin synthesis and hydrolysis,and chitin degradation is mainly done by chitinase,analysis of different periods and tissue in the tick(Rhipicephalus linnaei)chitinase RLCht1 sequence expression profile,in vitro heterologous expression protein and verify the activity of chitinase,for the tick and other vector biological control of reference were carried out,so as to provide a new target for developing safe and efficient biological tick.[Method]Different tissue samples of different period ticks and female blood-fed ticks were collected for differential expression analysis.Based on the putative chitinase sequence screened from the transcriptome of Rhipicephalus sanguineus,the recombinant prokaryotic plasmid was amplified and constructed in Rhipicephalus linnaei,and the E.coli system was used to express the chitinase protein RLCht1.After the inclusion body treatment,the protein was purified by nickel column and the RLCht1 protein domain was predicted.After purification using a nickel column,RLCht1 protein domain structure was predicted.The catalytic activity size and mode of action of purified chitinase RLCht1 were tested by a catalytic reaction with three different fluorescent substrates.[Result]The RLCht1 gene was expressed at different stages of satiety and in different tissues of satiated female adult ticks.The expression of RLCht1 gene in the satiated tick stage was extremely significantly higher than that in the egg stage(P＜0.01)and other stages(P＜0.05),and had a higher expression in the midgut of satiated female adult ticks.The recombinant plasmid RLCht1-pET-28a was constructed and the RLCht1 protein was successfully expressed.After inclusion body treatment,the purified RLCht1 protein was obtained by elution at a concentration of 150 mmol/L imidazole.The protein prediction results showed that RLCht1 belonged to the GH18 family chitinase.Fluorescent substrate enzyme activity detection showed that RLCht1 inclusion body dissolution solution,refolded unpurified prokaryotic protein,and refolded purified concentrated prokaryotic protein had chitin exonuclease activity of 0.078 and 0.350 U/mL,and β-N-acetylglucosaminidase activity of 0.034 U/mL,respectively.[Conclusion]RLCht1 might be related to the activity of ticks,affect life activities related to immunity,digestion and humoral regulation.The study successfully expressed chitinase protein for the first time in the Rhipicephalus linnaei,demonstrating that the purified RLCht1 protein had chitin catalytic function and provided a theoretical basis for the application of chitinase RLCht1 protein in the development of ticks in the future.
Rhipicephalus linnaeienzyme activity assayexpression spectrumprokaryotic expressioninclusion body
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