Study On Relationship Between Serine Proteases And Resistance In Tetranychus Cinnabarinu(Boisduval)

The carmine spider mite Tetranychus cinnabarinus is a harm serious and widely distributed plant pests in agricultural production,and it is distributed all over the world.It have happened in parts of China including South China,northwest China,southwest China,Hubei province and et,al.As many as 1000 species of host plants,which has more than 100 kinds of economic crops.Mite can easily develop acaricide resistance because of its high reproductive potential,short life cycle and so on.Serine proteases are a kind of important hydrolase in insects,and it play a important role in keeping normal physical activity.Serine proteases are involved in insect body's digestion,embryonic development,tissue reconstruction and so on.what wo?ld be a connection between the resistance of Tetranychus cinnabarinus and Serine proteases.In this study,a resistance strain of T.cinnabarinus against fenpropathrin or abamectin was selected and the connection between the resistance of Tetranychus cinnabarinus and serine proteases was investigated.The main results were as follows: 1.One gene of serine protease was cloned based on the genomes of T.urticae,which was named as SP-900.The gene sequence of nucleotides was 897 bp,and successfully translated it into 298 amino acid sequence.The result of sequence analysis shows that it is a serine protease gene.2.The expression levels of SP-900 gene in SS,FeR and AbR strains were detected via the comparative quantitative real-time PCR(qPCR).The results showed that the expression levels of SP-900 gene were significantly different strains.The expression levels of SP-900 gene in FeR and AbR strain were 2.45,and 5.54-fold when compared with SS strain.The expression of SP-900 at different time after fenpropathrin-treatment or abamectin-treatment were detected.From the results,we find that there is no significant differences between fenpropathrin-treatment and control after 6h,and 24 h in SS strain.The relative expression levels of SP-900 in the SS strain increased to 3.05-fold compared with control after 12 h fenpropathrin induction..The expression levels in the FeR strain increased to 2.77-and 2.65-fold compared with control after 6 h,and 12 h fenpropathrin induction,respectively.The expression levels in the SS strain and AbR strain increased to 2.42-and 2.25-fold compared with control after 12 h abamectin induction,respectively.These res?lts suggested that the SP-900 gene can be induced in SS,FeR,and AbR strains by fenpropathrin or abamectin.The serine proteseas may take part in mites,physiological and biochemical activities when dealing with the negative influence of drugs.3.The determination of serine proteases activity showed that the SQR activities were 3.0024nmol/ug/min,5.2634 nmol/ug/min,and 6.2774 nmol/ug/min in SS,FeR,and AbR strains,respectively.The activitiy of serine proteseas were 1.75-and 2.09-fold in FeR and AbR strains when compared with SS strains,respectively.The determination of serine proteases activity after serine protease inhibitors(APMSF)-treatment showed that the inhibitor can inhibit the enzyme activity by the method of modified RCV,the enzyme activities were suppressed by 38.4%,24.8%,and 21.9% witn control in SS,FeR and AbR strains,respectively,and mites became more sensitive to drugs.The res?lts suggested that with declining serine proteases,activity in the carmine spider mite,the sensitivity to the drugs rised.4.To clarify the specific influence of the over-expressed serine protease genes on the susceptibility to fenpropathrin and abamectin in T.cinnabarinus,a RNAi via dsRNA feeding experiment was performed.The expression of the target serine protease gene(SP-900)decreased by 49.19%,51.74% and 53.69% with control in SS,FeR and AbR strains,and the enzyme activities were suppressed by 28.6%,18.2% and 23.5% witn control in SS,FeR and AbR strains,respectively.The LC50 of fenpropathrin and abamectin decreased down 174 ppm and 0.089 ppm in SS strain after RNAi.The LC50 of fenpropathrin decreased down 14234 ppm in FeR strain,the LC50 of abamectin decreased down 0.211 ppm in AbR strain.These results suggested that feeding of SP-900 gene dsRNA can decrease the expression levels of SP-900 gene in SS,FeR and AbR strains,and lead to the enzyme activity decreased,the sensitivity to the drugs rised.5.The experiment of fenpropathrin or abamectin added to Crude enzyme which showed that the enzyme activities have no changed in SS strain.The serine proteases not to participate in the activities of catabolism for drugs.The ATP contents in SS,FeR and AbR strains of T.cinnabarinus were measured by HPLC.The results showed that the levels of ATP were no significant difference among strains.The FeR and AbR strains need more ATP to stronger detoxification metabolic activity than in SS strains.The resistance fitness cost happened in resistant strains such as Fertility dropped,the mites need less ATP in this part,so the levels of ATP were no significant difference among strains.