Zhang Chuanxi

CLONING AND COMPARISON OF THE GENES ENCODING PREPROAPAMIN FROM THE VENOM OF 2 HONEYBEE AND 4 WASP SPECIES

Abstract Preproapamin genes were amplified by RT‐PCR from total RNA from the venom glands of 2 honeybee species, Apis mellifera, A. cerana cerana, and 4 wasp species, Vespa magnifica, V. velutina nigrothorax and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM® ‐T easy vector and the nucleotide sequences analyzed. The six fragments were all 141 bp in length and contained an ORF coding the precursor of apamin. The apamin precursors of V. magnifica, V. velutina nigrothorax and P. hebraeus had 95% and 93% similarity with that of A. melliera in nucleotide and amino acid sequences, respectively. That of Vespula maculifrons was identical to that of A. mellifera in nucleotide and amino acid sequences. Apamin precursors of V. magnifica, P. hebraeus and V. velutina nigrothorax also had the same nucleotide sequences. The nucleotide sequences of preproapamin genes from the Chinese honeybee, A. cerana cerana and 4 wasp species are described for the first time. A notable discovery was that the wasps species had exactly same apamins as the honeybees despite the fact they belong to different insect families.

THE CLONING, SEQUENCING AND EXPRESSION OF THE LdMNPV POLYHEDRIN GENE

Abstract  LdMNPV ‐ NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of MMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the MMNPV‐NEFU isolate and a Canada strain, MMNPV‐G differed in 5 bases. The polyhedrin gene of the LdMNPV‐NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV‐G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV‐NEFU isolate and by CAC in the MMNPV‐G isolate. The MMNPV polyhedrin gene was expressed in E. coli BL21 (DE3) by the pT7–7 plasmid vector.

CLONING AND SEQUENCING OF GENE ENCODING HYALURONIDASE FROM THE VENOM OF APIS CERANA CERANA

Abstract The venomous hyaluronidase (Hya) gene of Chinese honey bee, Apis cerana cerana, was amplified by RT‐PCR from total RNA of venom glands of the worker bees. The full length of its nucleotide is 1164 bp encoding a 387 amino acid polypeptide with predicted molecular weight of 42.6 kD. The alignment of AcHya amino acid sequence with other 6 Hyas shows that AcHya is most closely related to the Hya of European honey bee, A. mellifera, with 91% amino acid identity. It also shares homology with Hya of Dolichovespul amacidata, Polistes annularis, Vespula vulgaris, Lutzomia longipalpis and Homo sapiens (sperm), with 54%., 52%., 46%, 27% and 20% amino acid identity, respectively. A phylogenetic tree of those hyaluronidases was drawn by using GENETYX program, the conservation, the relationship between molecular structure and function of 7 hyaluronidases as above was compared and analysed.

ANALYSIS OF HELICOVERPA ARMIGERA SINGLE NUCLEOPOLYHEDROVIRUS IMMEDIATE EARLY 1(IE-1) GENE

Abstract A 6.12 kb Xbal‐H fragment of the Helicoverpa armigem single nucleopolyhedrovirus (HaSNPV) gemone was cloned and the complete sequence of this fragment was sequenced by random sequencing method. Sequence comparison and analysis revealed an ORF13 which was homologous to ie‐1 of Auiographa California nucleopolyhedrovirus (AcMNPV). The homologous encoding gene is ie‐1. The total length of the encoding region of HaSNPV gene was 1986 bp and was predicted to encode 661 amino acid protein(IE‐1) with molecular weight of 76.5 kD. The alingment of putative HaSNPV IE‐1 amino acid sequence with those of other 9 reported baculoviruses IE‐Is showed that the HaSNPV IE‐1 was most closely related to Helicoverpa zea nucleopolyhedrovirus (HzNPV) IE‐1, with 97% amino acid identidy. But it showed a low degree of sequence similarity to those of AcMNPV, Bombyx mori nucleopolyhedrovirus (BmNPV), Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV), Lymantria dispar nucleopolyhedrovirus (LdMNPV), Orgyia pseudotsugata nucleopolyhedrovirus (OpMNPV), Spodoptera exigua nucleopolyhedrovirus (SeMNPV), Plutella xylostella granulovirus(PxGV) and Xestia c‐nigrum granulovirus (XcGV), with 23%, 23%, 23%, 25%, 23%, 14%, 27% and 7% amino acid identity, respectively. A phylogenetic tree of ten baculoviruses IE‐1 was also given.