2 cdna ~ Bt ~ Zhao 1. 2 CrylC 3l 8 Bt First-Strand Synthesis System Invitrogen Bt 9 ~ 11 PCR DNA N Invitrogen peasy-t1 aminopept

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1 Chinese Bulletin of Entomology ~ 274 檻檻檻檻檻檻檻檻檻檻檻檻檻檻 cdna * Cloning and sequence analysis of alkaline phosphatase gene in the diamondback mothplutella xylostella. LIU Jie 1 2 WEN Li-Zhang 1 WANG Shao-Li 2 WU Qing-Jun 3 ZHANG You-Jun 2 1. Hunan Agricultural University College of Bio-safety Sciences and TechnologyHunan Agricultural UniversityChangsha China 2. Institute of Vegetables and FlowersChinese Academy of Agricultural SciencesBeijing China Abstract Alkaline phosphatase genes were cloned from the resistant and susceptible strains of Plutella xylostella L. through RT-PCR technique using degenerate primers and sequenced. The cdna length was 403 bp and the sequence was confirmed to be the alkaline phosphatase gene by blast alignment BLAST. The nucleotide similarity of the resistant and susceptible strains was 99. 5% and the amino acids identity was 99%. Compared with the susceptible strain18 nucleotides were changed in the resistant strainresulting in one amino acid changed V to T. These results are helpful for further study of the full sequence and function of alkaline phosphatase gene in P. xylostella. Key words Plutella xylostellaalkaline phosphatasecloningsequence analysis DNAman RT-PCR Plutella xylostella L. Bt 403 bp blast 18 1 Plutella xylostella L Tabashnik 10 1 Bt 5 Ferre Bacillus thuringiensisbt 95% 2 3 Bt 6 Bt Bt * CB AA10Z421 niuniu @ 163. com 2 4 Bt zhangyj@ mail. caas. net. cn

2 2 cdna ~ Bt ~ Zhao 1. 2 CrylC 3l 8 Bt First-Strand Synthesis System Invitrogen Bt 9 ~ 11 PCR DNA N Invitrogen peasy-t1 aminopeptidasesapn DNA Marker cadherin-like protein alkaline phosphatases ALP glycolipid ~ Bt Cry1A 15 Jurat- Fuentes 0. 1% TritonX-100 Heliothis virescens Cry1Ac Bt 5 ~ 7 16 m-alp 4. 8 cm Bt h reverse-transcription polymerase chain reaction 72 h LC 50 RT-PCR 10% cdna Cry1Ac Bombyx Bt moriab Helicoverpa armigera TRIzol SuperScript TM Ⅲ Tabashnik s EU Aedes aegypti XM DNAMAN 6. 0 Alp-f S GGDGGGCGCATCGACCACGCGCA-3 Alp-r 5 -ACGTCDTCKCCGCCGTGCGTC- 25 ± 1 60% ~ TC-3 70% = 16 h 8 h RNA cdna R Cry1Ac TRIzol 300 RNA cdna 50 μl 1 μg RNA 50 Cry1Ac 50 2 μmol / Oligo dt 16 1 μl 5 M-MLV buffer 10 μl 2. 5 mmol / L each dntp 5 μl 40 U / μl Rnase Inhibitor 0. 5 μl 200 U / μl RTase M-

272 Chinese Bulletin of Entomology 47 MLV 1 μl 42 1 h 70 15 min M-MLV 1. 3. 4 RT-PCR RT-PCR 94 6 min 35 94 30 s 50. 6 SilverBeads peasy-t1 TOP-10 10 PCR 2.

3 272 Chinese Bulletin of Entomology 47 MLV 1 μl 42 1 h min M-MLV RT-PCR RT-PCR 94 6 min s SilverBeads peasy-t1 TOP PCR 2. 2 RT-PCR Alp-f Alp-r RT-PCR ddh 2 O bp PCR 400 bp Cry1Ac LC μg / ml μg / ml s 72 1 min 72 1 Cry1Ac 10 min 1% 95% μg / ml S ~ ± R ~ ± RT-PCR PCR ALP RT-PCR PCR M1 Marker 1 S1 R1 R2 SZ1 RZ1 RZ2 D1 D % ALP 93% 86% 79% 403 bp blast ALP 69% m-alp 93% s-alp

4 2 cdna ALP ALP 23 Cry1Ac 2 - cdna RACE 3 1 Talekar N. S. Shelten A. M. Biologyecology and - management of diamondback moth. Annu. Rev. Entomol ~ Soberon M. Pardo-Lopez L. Lopez I. et al. Engineering modified Bt toxins to counter insect resistance. Science 3 m-alpcdna bp ALP 481 Cry1Ac 18 1 V T Bt Bt 1 RNAi Bt Bt Bt Bt ~ Romeis J. Meissle M. Bigler F. Transgenic crops expressing Bacillus thuringiens is toxins and biological

5 274 Chinese Bulletin of Entomology 47 control. Nature Biotechnol ~ Romeis J. Bartsch D. Bigler F. et al. Assessment of risk of insect-resistant transgenic crops to non target arthropods. Nature Biotechnol ~ Tabashnik B. E. Cushing N. L. Finson N. et al. Field development of resistance to Bacillus thuringiensis in diamondback month. J. Econ. Entomol ~ Cry1Ac-receptor alkaline phosphatase in susceptible and 6 Georghiou G. P. Talor C. E. Proceedings of ⅩⅤth resistant Heliothis virescens larvae. Eur. J. Biochem International Congress of Entomology. Washington D. C ~ College Park ~ Heckel D. G. Gahan L. J. Tabashnik B. E. et al. The 7. diversity of Bt resistance genes in species of Lepidoptera. J ~ 244. Invertebr. Pathol ~ Zhao J. Z. Collins H. L. Tang J. D. et al. Development 18 Pigott C. R. Ellar D. J. Role of receptors in Bacillus and characterization of diamondback moth resistance to thuringiensis crystal toxin activity. Microbiol. Mol. Biol. R. transgenic broccoli expressing high levels of CrylC. Appl ~ 281. Environ. Microbiol ~ Tabashnik B. E. Cushing N. L. Johnson M. W. 9 Tabashnik B. E. Gassmann A. J. Crowder D. W. et al. Insect resistance to Bt crops evidence versus theory. Nature Biotechon ~ 202. Diamondback moth resistance to insecticides in Hawaii intraisland variation and cross-resistance. J. Econom. Entomol ~ Ferre J. Van Rie J. Biochemistry and genetics of insect 20 Chen Q. X. Yan S. X. Substrate specificity of Amphioxus resistance to Bacillus thuringiensis. Annu. Rev. Entomol. ALPase and on the influence of some effectors on the enzyme ~ Jurat-Fuentes J. L. Adang M. J. Cry toxin mode of action in susceptible and resistant Heliothis virescens larvae. J. Invertebr. Pathol ~ Zhu Y. C. Kramer K. J. Oppert B. et al. Cdnas of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins. Insect Biochem. Mol. Biol ~ Gahan L. J. Gould F. Heckel D. G. Identification of a gene associated with Bt resistance in Heliothis virescens. Science ~ Xu X. J. Yu L. Y. Wu Y. D. Disruption of acadherin gene associated with resistance to Cry1Ac-endotoxin of Bacillus thuringiensis in Helicoverpa armiqera. Appl. Environ. Microbiol ~ Griffitts J. S. Whitacra J. L. Stevens D. E. et al. Bt toxin resistance from loss of a putative carbohydrate-modifying enzyme. Science ~ Jurat-Fuentes J. L. Adang M. J. Characterization of a J. Xiamen Univ. Natural Scienc ~ Rao N. M. Nagaraj R. Anomalous stimulation of Escherichia coli alkaline phosphatase activity in guanidiniun chloride. J. Biol. Chem Besman M. Coleman J. E. Isozyme of bovine intestinal alkaline phosphatase. J. Biol. Chem ~ Misbahuddin R. S. Ehteshamuddin S. Effect of phosphamidon on the alkaline phosphatase activity in the haemalymph of sap feeding insect pests Aspongopus janus Chrysocoris stollii and Dysdercus cingulatus. Environ ~ 325. Ecol.

3 461 DNA lx PDV PDV 2 10% ~ 15% PDV PDV 1 PDV PDV PDV Tagashira PDV d 5 Co

3 461 DNA lx PDV PDV 2 10% ~ 15% PDV PDV 1 PDV PDV PDV Tagashira PDV d 5 Co Chinese Bulletin of Entomology 2010 47 3 460 ~ 466 檻檻檻檻檻檻檻檻檻檻檻檻檻檻 * 450002 Parasitic effects on host Plutella xylostella testes by two endoparasitoids. HE Fan BAI Su-Fen LI Xin CHAO Yun-Fei College of