| 1. Molecular cloning and sequence analysis of GABA receptor genes from Plutella xylostellaTwo GABA receptor alpha subunit genes were cloned from Plutella xylostella using RT-PCR and RACE techniques, designated as PxGABARal (GenBank accession no. FJ665608) and PxGABARa2 (FJ665610), which have open reading frame of 1458 and 1563 nucleotides respectively encoding 485 and 520 amino acids. The two subunit genes both have four conserved transmembrane domains and cysteines. At the orthologous A302S mutation position of D. melanogaster Rdl, the PxGABARal had a 282A, however, the PxGABARa2 had a 285S in susceptible Roth strain. Pair-wise comparison indicated that the deduced PxGABARal amino acid sequence shared high identity with HvGABARa1 of Heliothis virescens (AF006192,95%), HvGABARa2 of H. virescens (AJ224513,85%), DmRdl of D. melanogaster (AAF50311,84%), PxGABARa2 of Plutella xylostella (FJ665610,83%), BmGABARal of B. mori (EF521821,84%). The deduced PxGABARa2 amino acid sequence shared high identity with HvGABARa2 of H. virescens (AJ224513, 91%), SeGABARa2 of S. exigua (ABQ45398,87%), BmGABARal of B. mori (EF521821, 84%), HvGABARal of H. virescens (AF006192,83%), DmRdl of D. melanogaster (AAF50311,77%). Two splicing form at exon 3 and one splicing form at exon 6 were found in both subunits.2. Genomic structure and genetic mapping of GABA receptor subunits in Plutella xylostellaGenomic DNA sequences, with exception of the first intron, of PxGABARal and PxGABARa2 were obtained by using genome walking and routine PCR technique with serial primers designed according to the known cDNA sequence of them. The coding regions of both two subunit genes comprised nine introns, the intron-exon boundary sequences of all introns followed the GT-AG rule, both of two subunits shared identical intron insertion site, and intron phase were highly consistant with each other, which suggest that these two genes may be derived from a recent gene-duplication event. In order to determine whether these two genes are located on the Z chromosome, two informative DNA markers were exploid to test genotypes of F1 progeny from single-paire crosses between female SZ-Diel(ZSerW) and male Roth moths(ZAlaZAla). As for PxGABARα1, among 66 F1 progeny,33 female F1 were hemizygous for A282, and 33 male F1 were heterozygous for A/S 282, these results indicat that PxGABARα1 locus is located on the Z chromosome. For PxGABARα2, all 15 tested female F1 were hemizygous for the long intron fragment, while all 19 tested male F1 were heterozygous having both long and short inron fragments.That mean that the PxGABARα2 locus is also located on the Z chromosome.3. Relationship between A282S mutation of PxGABARα1 subunit and dieldrin and fipronil resistance in Plutella xylostellaIn order to clarify the role of A282S mutation of PxGABARα1 for resistance to dieldrin and fipronil in P. xylostella, two P. xylostella strains with homozygous A282S mutation, which are resistant to fipronil and dieldrin respectively, were established by single-paire crosses, namely SZ-FSer and SZ-DielSer, and the near isogenic line strain resitant to fipronil SZ-FAla as well. Comparing with susceptible Roth strain and SZ strain, SZ-FSer shown 208.43-and 182.75 fold resistance to fipronil, only exhibited low level of cross-resistance to dieldrin, endosulfan, lindan comparing with control strain SZ, namely 6.4-1.89-,5.81-fold, respectively, no cross-resistance to abamectin and spinosad. SZ-DielSer strain developed 9.3-and 6.52 fold resistance to dieldrin comparing with Roth and SZ strain, and 10.91-,6.07-,8.95 fold cross-resistance to fipronil, endofulfan and linden, respectively, no cross-resistance to abamectin and spinosad. SZ-FAla exhibited 101.20-and 88.73 fold resistance to fipronil comparing with Roth and SZ strain, respectivety, no cross-resistance to dieldrin and endosulfan.SZ-FSer, SZ-DielSer, SZ-FAla showed 6.4-,6.52-and 1.34 fold resistance to dieldrin comparing with control strain SZ. SZ-FSer, SZ-DielSer had 4.77-,4.86 fold resistance to dieldrin comparing with SZ-FAla strain, while SZ-FSer only exihibited 2.06 fold resistance to fipronil, which indicated that the A282S mutation in PxGABARα1 is a major factor for dieldrin resistance and minor factor for fipronil resistance in P. xylostella.Genotype were identified for the survival female and male individual after treating with various insecticides towards offspring of reciprocal crosses between the dieldrin resistant strain SZ-DielSer and the susceptible strain Roth. The survival percentage of F1 with ZSerZAla, ZAlaW, ZSer ZAla and ZSerW toward 1.5ppm fipronil are 8.5%,6.5%,9%,10.1%, respectively, after treated with 600ppm dieldrin, there are 6.5%, all death,5%,16.5%, respectively, treated with 1,500ppm endosulfan, there are 11%,2.5%,13.5%,15.5%, respectively, these results indicate again that the A282S mutation in PxGABARal is a major factor for dieldrin resistance and minor factor for fipronil and endosulfan resistance in P. xylostella, and dieldrin resistance in SZ-DielSer strain is linked to the Z chromosome.4. Heterologous expression in insect cell of carboxylesterase genes from Helicoverpa armigeraEntry clones were obtained by using Gateway technique through recombination of entry vector with carboxylesterase CCE001a, CCE001c, CCE001d, CCE001i and CCE001j from YG and YGF strain of Helicoverpa armigera. Analysis for all deduced amino acid sequences showed they all contained the amino acid motifs thought to be necessary for catalytic activity, including conserved cysteines, an oxyanion hole (GG), and the catalytic triad consisting of Ser, Glu and His, polymorphsm of amino acid were found at several position, none of the H. armigera CCEs tested had a mutation equivalent to G137D but two of them (CCE001c and CCE001i) had a L residue equivalent to W251L. A four amino acid residues insertion nearby C-terminal was found in CCE001i from YG strain compared with that of YGF strain. All recombinant viruses were transfected in insect cell Sf9 successfully, preliminary activity assays were conducted with native PAGE staining with 1-NA, With the exception of CCEOOli, the carboxylesterases tested showed 1-NA hydrolysis activity. A high titre virus stock was then prepared for use in the expression experiments, with viral titres determined by quantitative PCR, after that all H. armigera CCE expressed in insect cell Sf9.5. Hydrolysis activities of Helicoverpa armigera carboxylesterase expressed in vitro toward organophosphate and esterase substratesHydrolysis activities of organophosphates and titration of esterase active sites for all expressed protein were measured with two fluorescent organophosphate substrates, dEUP and dMUP. None of the expressed carboxylesterases show the huge increase in OP hydrolysis seen in E3. Only some of them show increased OP hydrolysis activity compared with E3 WT. CCEOOld from both YG and YGF strain yielded similar kcat values for both dEUP and dMUP, however the other four H. armigera CCEs from YG and YGF showed a strong preference for dMUP, with values 1.8-to 4.6 fold higher, this characteriazation of preference for dMUP is also seen with the W251L mutant of E3, two of them(CCE001c and CCE001i) had a L residue equivalent to W251L, but the other two(CCE001a and CCE001j) had a F residue equivalent to W251L, which indicated that a L residue at site equivalent to W251L is not a necessary factor for the dMUP preference.The recombinant protein of H. armigera CCEs showed preference for a-NA over p-NPA as substrate. Three of five expressed H. armigera CCEs (001a,001j,001d) exhibited higher hydrolysis activity toward a-NA than that of the other two (001c,001i), the three CCEs (001a,001j,001d) had a L residue equivalent to W251L, F for that of 001c and 001i, so the differential hydrolysis activity toward a-NA may related to the differential amino acid residue equivalent to W251L. No significant difference was found in activity toward a-NA for 001a and 001j between YG and YGF strain, while 001c and 001d showed 2-and 1.5-fold higher in YGF strain, repectively. For substrate p-NPA,001c and 001j had significant differenc between two strains.001a,001d,001j exhibited a higher affinity toward a-NA, they would therefore play an important role in pyrethroid resistance via sequestration mechanism.6. Hydrolysis activities of Helicoverpa armigera carboxylesterase expressed in vitro toward pyrethroids and their fluorogenic analogsIn order to learn the potency of expressed H. armigera CCEs to hydrolyse pyrethroids, hydrolysis assay for expressed H. armigera CCEs were conducted by using all stereoismers of cypermethrin and fenvalerate as well as their corresponding fluorogenic analogs. Differential stereoselectivity existed in cypermethrin analogs, which showed preference for trans over cis and 1(S) over 1(R). In general, the stereoisomer 1(S)trans-a(S) and 1 (R)trans-a(R) displayed higher activities than other stereoisomers for all expressed H. armigera CCEs. For fenvalerate analogs, the acidic moiety with the 2R form showed higher activity for all expressed H. armigera CCEs with the exception of 001 i.001a,001d,001j significantly improved activities for all fluorogenic analogs of fenvalerate comparing with that of E3 WT,001c showed only low activities against two of four fluorogenic analogs of fenvalerate. Similarly, the same pattern which is preference for trans over cis and 1(S) over 1 (R) hydrolysed by expressed H. armigera CCES also seen in all isomers of cypermethrin, isomers of 1 (S)trans-a-(S) and 1(R)trans-a-(R) exhibited the highest activities for all expressed H. armigera CCES with exception of 001 i, however, activities of other isomers increased to some extent comparing with their corresponding fluorogenic analogs. Hydrolysis activities to esfenvalerate ranged from 0.3-to 5.39pmol/min/pmol. Expressed H. armigera CCEs exhibited pyrethroid hydrolysis activity in this study, which showed over-expression on mRNA level in previous report, could be an important factor for fenvalerate resistance in H. armigera. |
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