Effects Of Abamectin And Heat Stresses On The Expression Of GABA And GABA Receptor In Tetranychus Cinnabarinus

The carmine spider mite,Tetranychus cinnabarinus(Boisduval),one of the most important pests on cotton,vegetables and other crops,is widely distributed in China.Because of its high fecundity and short generation time,the mite can form rapidly population 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.The thesis was designed to detect and compare the differences of GABA and GABAR content among susceptible strain(SS),abmectin-resistance(AbR) and high temperature resistance strains(HR) of T.cinnabarinus,and discussed the biochemistry and molecular resistance mechanism of T.cinnabarinus to abamectin and heat stresses,in order to reveal the thorough mechanism of superior fitness of abamectin-resistant strains under high temperature condition,and to provide a theoretical basis for Integrated Resistance Management(IRM).This work was supported by grants from the National Natural Science Foundation of China(No.30600059),and the main results were as the following.1.The method for measuring the content of GABA,using Phenylisothiocyanate(PITC) as pre-column derivatization reagent with high-performance liquid chromatographic(HPLC) UV detection,was established.The method exhibited good retention time(4.29min) and peak area reproducibility for GABA-PITC.The recoveries of GABA were among the the range of 98.53%-100.60%.2.The GABA content in T.cinnabarinus was measured by HPLC,the content in AbR strain of T. cinnabarinus was significantly higher than that in SS strain of T.cinnabarinus(1.39 fold,P＜0.05), and the GABA content in abamectin-resistance individuals selected from SS with abamectin LC95 of SS(SS-A) and SS-AF₁(the F1 generation of SS-A) strains were also significantly higher than that in SS(1.41-and 1.52-fold,respectively).The significantly higher expression level was observed in the SS strain 8 hours later of being treated with abamectin(0.018 mg/L) when compared that with no treatment(the content of GABA increased 52%),whereas the expression level in AbR strain did not change significantly after being treated with abamectin.The GABA content of HR strain was significantly lower than that of SS(about 73%of that in SS) and the content of GABA in SS and HR were significantly risen an hour later of 40℃heat-shock(increased 27.2%and 20.6%, respectively).We speculate the changes of GABA content is involved in the resistance to abamectin and high temperature in T.cinnabarinus.3.A comparative study on crawling speed of T.cinnabarinus with the high and normal GABA content was carried on to verify the inhibitory function of GABA.We found that the crawling speeds of AR,SS-A and SS-AF₁ were significantly slower than that of SS(about 62.3%,70.8%and 76.4% of the speed of SS,respeetively).The significantly slower speed was also observed in the SS 8 hours later of being treated with abamectin when compared that with no treatment,however the speed in AbR strain did not change significantly after being treated with abamectin.There was no significance difference between the speeds of SS and HR strains,whereas their movement slowed down obviously after being treated with high temperature(40℃).4.We cloned a novel member of the invertebrate GABA-gated chloride channel gene family from T.cinnabarinus,through rapid amplification of cDNA ends(RACE) and polymerase chain reaction using primers based on the 5' and 3' ends of the mRNA(Genbank EU362111 named as Rdl^Tc).The complete amino acid sequence of Rdl^Tc deduced from the cDNA is 1730 residues with the putative signal peptide consisted of 21 residues with a theoretical molecular weight 41.1 kDa and an isoelectric point(pI) of 9.25.The 5' and 3' untranslated regions(UTR) consist of 28 and 555 bp, and there is a 16 bp poly A tail in 3'untranslated region.The predicted GABA receptor protein shared 72 and 71%identity with other GABA receptors proteins isolated from Tetranychus urticae and Drosophila mlanogaster,at amino acid level,respectively.Analysis of the amino acid sequence of Rdl^Tc indicated it contains a putative polydenylation signal AATAAA,highly conserved dicysteine-loop(Cys-loop) and four transmembrane domains(TM1,TM2,TM3,and TM4).Online software(http://www.cbs.dtu.dk/serviees/NetNGlyc,http://www.cbs.dtu.dk/serviees/NetPhos) was used to analysis the N-glycosylation sites and phosphorylation sites.The highly conserved N-glycosylation sties(N92 and N133 in Rdl^Tc) and highly conserved phosphrylation sites(187Y, 341S,346Y and 362Y in Rdl^Tc) are all occurred in other insect and mite species.While N-glycosylation sties(60N in Rdl^Tc) and phosphrylation sites(48S and 339S in Rdl^Tc) are mainly confined in mites,which suggesting these motifs are species-specific.The phylogenetic tree shows that acarus is well separated from insect.Among acarus,the Tetranychidae and Ixodidae are well separated.Among insect,Diptera,Hymenoptera,Homoptera and Lepidoptera are well separated from each other,but under order level,family can't be well separated,such as H.virescens,B.mori, P.xylostella,and S.exigua in Lepidoptera.Therefore,our results suggested that the sequences of GABAR are well separated on order level and might be used to construct phylogenetic tree on this level.we also compared the cDNA sequences of the Rdl^Tc between SS and AbR strain of T. cinnabarinus and found that there was no mutation in Rdl^Tc in abamectin-resistant strain seleted in laboratory.5.The mRNA expression level of Rdl^Tc was detected by the method of SYBR Green I dye-based technology with theβ-actin as reference gene.The expression level of the GABA receptor gene in AbR strain was significant lower than that in the SS strain.The significant lower expression level was observed in SS strain 8 hours later after being treated with abamectin when compared that with no treatment,whereas the expression level in AbR strain did not change significantly after being treated with abamectin.The expression level of the GABAR in HR was significant lower than that in SS.The significant lower expression level was also observed in SS after heat shock,however there was almost no change after being treated with high temperature in HR.The low expression level of GABA receptor is involved in the resinstance to abamectin and high temperature in T.cinnabarinus.