Molecular cloning and characterization of acid phosphatase in venom of the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae).
The present study describes cDNA cloning, sequencing, enzyme activity determination and localization, and mRNA expression of acid phosphatase in the venom apparatus of an endoparasitoid, Pteromalus puparum. This is the first report of cloning a venom acid phosphatase gene which has been described in parasitoid wasps. The cDNA consisted of 1378bp with 1215bp open reading frame and encoded a sequence of 405 amino acids. A 23 residues N-terminal signal peptide was followed by a short 15 residues (25-39) histidine acid phosphatases phosphohistidine signature and a long 302 residues (24-325) acid phosphatase family domain. The deduced amino acid sequence shared 32-88% identity to its counterparts from other insects. Enzyme activity was measured by using p-nitrophenyl phosphate (p-NPP) as substrate, and a high level of acid phosphatase activity in venom was detected. Optimal pH and temperature for this enzyme activity was 4.8 and 45 degrees C, respectively. Ultracytochemical analyses further revealed that strong enzyme activity was located in the nuclei and secretory vesicles of the venom gland secretory cells. Expression of the acid phosphatase gene was observed to be regulated at different developmental stages by RT-PCR analysis as it expressed immediately with low abundance after adult emergence, then increased to the high level at 2-4 days, followed by a drop to the low abundance after 4 days. Compared to the mRNA expression, a time-course-related enzyme activity in an individual venom apparatus was also found.
Zhu
Jia-ying
JY
State Key Laboratory of Rice Biology, Institute of Insect Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.
Ye
Gong-yin
GY
Hu
Cui
C
D000135:Acid Phosphatase / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D016366:Open Reading Frames; D009943:Organophosphorus Compounds / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014862:Wasp Venoms / Q000201:enzymology* / Q000235:genetics; D014863:Wasps / Q000201:enzymology* / Q000235:genetics
D019476:Insect Proteins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012333:RNA, Messenger; D014862:Wasp Venoms; C008644:nitrophenylphosphate; D000135:Acid Phosphatase