Esterase Gene CDNA Cloning Of Tetranychus Cinnabarinus (Boisduval) And Detection Of Its MRNA Expression

The carmine spider mite,Tetranychus cinnabarinus(Boisduvat),one of the most important pests on cotton,vegetables and other crops,is widely distributed in China.Because of high fecundity and short generation time,the mite can rapid formation of a certain number of stocks in a short period of time,develop resistance easily and thus it is difficult to prevent and control this mite.Esterases belong to serine hydrolase family able to hydrolysis carboxylic acid ester and phosphate bond.As a large class of enzymes that metabolize a variety of pesticides,esterases play important roles in priority hydrolysis of water-soluble short-chain esters of the ester.The research of esterase gene cloning is very active in foreign and relative behindhand in domestic.The full-length cDNA of two esterases genes TCE1 and TCE2 from carmine spider mite sensitive strains were obtained with RACE technology,and the mRNA expression differences of the two genes in different developmental period(egg,protonymph,nymphae and adults),different strains(abamectin-resistant,AbR;fenpropathrin-resistant,FeR;omethoate-resistant,OmR and sensitive strains,S)and abamectin-induced or not from carmine spider mite were also detected using real-time FQ-PCR technology,respectively.Besides,a preliminary study on inherent relation between the expression of the two genes and acaricide-resistant form in molecular level were carried out.This work was supported by grants from the National Natural Science Foundation of China(No. 30571239),and the key results were as follows.1.The full-length esterase genes cloning from T.cinnabarinusTwo full-length cDNA-encoding esterase genes,provisionally named TCE1 and TCE2 respectively,were cloned from T.cinnabarinus susceptible strains by the method of rapid amplification of cDNA ends(RACE).The complete amino acid sequence of TCE1 deduced from the cDNA is 1793 residues with the putative signal peptide consisted of 20 residues with a theoretical molecular weight 62.75kDa and an isoelectric point(pI)of 6.41.The 5' and 3' untranslated regions (UTR)consist of 26 and 67 bp,respectively.And the complete amino acid sequence of TCE2 deduced from the cDNA includes 2023 residues with the putative signal peptide consisted of 23 residues with a theoretical molecular weight of 63.14kDa and an isoelectric point(pI)of 5.70.The lengths of 5' and 3' UTR were 96 and 244 bp,respectively.2.Sequence analysis of T.cinnabarinus esterase genesThe deduced amino acid sequence of the two genes both had the Esterase-lipase CO esterase, conservative domain and a full N and C-terminal in the GenBank.There was an amino acid identity of 46%between the sequences of two genes from T.cinnabarinus.The TCE1 possesses 34%amino acid identity to Boophilus microplus esterase and the TCE2 had 35%amino acid reside identity to Nilaparvata lugens ace precursor.The predicted amino acid of the two genes both contained some typical residues of AChE(the three residues that putatively form the catalytic triad,the oxy-anino hole and the sequence GESAG) but there were lower identities between them and other insect AChE genes.Both the two esterase genes possess Carboxylesterases type-B signature of F-[GR]-Gx(4)-[LIVM]-x-[LIV]-xGxS-[STAG]G(S is the active site residue). The corresponding amino acid residue of TCE1 and TCE2 genes were FGGnpdqVtIfGeSAG and FGGdpnrVtLfGqSAG,respectively.Molecular phylogenetic tree of estesrases amino acid sequences from some representative insects and mites were constructed.The results showed that all esterase genes selected were classified into several groups:Insect TypeⅠAChE Gene,Insect TypeⅡAChE Gene,Nematode AChE Gene,Tick & Mite Esterase Gene and Insect CarE & Esterase Gene.T.cinnabarinus esterase genes and Boophilus microplus esterase gene belong to the same original group showing that the two genes from T.cinnabarinus were esterase genes.3.Detection of the esterase genes mRNA expression from T.cinnabarinusThe sequence of theβ-actin gene cDNA fragment of carmine spider mite was amplified by reverse transcript polymerase chain reaction(RT-PCR)with a pair of degenerate primers designed according to ticks and insects actin genes sequence and the target sequence contained 822 base pairs (bp)encoded 273 amino acid residues.Furthermore,a real-time FQ-PCR method based on SYBR GreenⅠdye was developed and the results of this study could provide basis forβ-actin used as a reference gene to analyze the T.cinnabarinus gene quantitatively.The mRNA expression levels of TCE1 and TCE2 genes were detected from egg,protonymph, nymph and adults of T.cinnabarinus through the method of SYBR GreenⅠdye-based technology with theβ-actin reference gene.The results showed that the mRNA expression levels of TCE1 gene from different stages were 0.02334,0.02163,0.02057 and 0.05130 times as those ofβ-actin gene, respectively;as for TCE2 gene the indexes were 0.0422,0.00636,0.03757 and 0.23351 times, respectively.The mRNA expression levels of two esterase genes in adults of T.cinnabarinus were significantly higher than those from other instars,and highest expression level was found for TCE2 gene.The mRNA relative expression level of the TCE1 and TCE2 genes were also detected from T. cinnabarinus AbR,FeR,OmR and S.The results showed that TCE1 gene mRNA expression of the three resistant strains were 0.725,1.810 and 2.087 times as S,respectively,and 0.882,1.490 and 1.703 times as S,respectively for mRNA expression of the TCE2 gene.The mRNA expression of two esterase genes from T.cinnabarinus FeR and OmR were significantly higher than those from AbR and S and they expressed relatively lower but no significantly in AbR compared to that in S. The mRNA expression changes in TCE1 gene were larger than those in TCE2 among different resistant strains.After induced by abamectin,the mRNA expression level of TCE1 gene from T.cinnabarinus significantly decreased to 0.32 times and TCE2 gene to 0.84times.