Spatial characteristics of soil enzyme activities and microbial community structure under different land uses in Chongming Island, China: geostatistical modelling and PCR-RAPD method.

Information about soil enzyme activity and soil DNA as indicators of how land use affects soil quality remains unknown for alluvial island. The purpose of this study was to investigate the effects of land use on enzyme activities and soil gene diversity in the Chongming Island, Shanghai, China, with the aim of providing further knowledge about the problems related to soil quality and its evolving spatial characteristic on the Chongming Island. The results indicate that land use causes an important change of organic matter, with either causing a small decrease or an increase in the absolute enzyme activity. With increased management intensity, there is an increase in the number of soils with high organic matter content and a decrease in the soils with low organic matter content. The effect on enzyme activity varies depending on the type of land use or management and the type of enzyme. The orders of the number of gene polymorphic bands under different land uses were as follows: agriculture soil>commercial soil>industrial soil>wetland. The complexities of the behaviors of the soil enzymes indicate that genetic analysis and biomarkers in conjunction with geostatistics method will be a powerful and accurate indicator to evaluate soil quality under land use.

Expression of the melittin gene of Apis cerana cerana in Escherichia coli.

A cDNA encoding melittin in Apis cerana cerana was obtained by PCR from the recombinant plasmid and cloned into the GST fusion expression vector pGEX-4T-2 for expression of the protein. The expressed protein of about 29 kDa was detected by Western blot and triple antibody sandwich ELISA, indicating that the recombinant protein is the fusion protein of GST-AccM. The expression conditions of GST-AccM fusion protein for Escherichia coli BL21 transformant were optimized. Thin layer scanning on the SDS-PAGE profiles of GST-AccM showed that the expressed protein accumulated up to about 15.2% of total protein of bacterial cells under the optimized expression condition. Purified and recovered recombinant melittin of A. cerana cerana showed bioactivity in activating rabbit platelets to aggregate.

Cloning and characterization analysis of the genes encoding precursor of mast cell degranulating peptide from 2 honeybee and 3 wasp species.

The precursors of mast cell degranulating peptide (MCDP) genes were amplified by RT-PCR from the total RNA of venom gland of two honeybee species, Apis mellifera ligustica, Apis cerana cerana, and three wasp species, Vespa magnifica, Vespa velutina nigrothorax and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM T-easy vector and the nucleotide sequences were analyzed. The length of five fragments was the same, it was 341 bp containing an ORF of 153 bp coding the precursor of MCDP and 188 bp 3' noncoding region. They have more than 90% homologues with each other in nucleotide sequences. The precursors of MCDP of A. cerana cerana, V. magnifica, V. velutina nigrothorax and P. hebraeus shared 96%, 100%, 94% and 98% homology with A. mellifera ligustica, respectively. The two species of wasps, V. magnifica and V. velutina nigrothorax, contained the same MCDP as A. mellifera ligustica, though they belong to different families with quite different biological properties, while A. cerana cerana contained the different MCDP in their venom as A. mellifera ligustica though they belong to the same genus. The fifth amino acid residue of MCDP in A. cerana cerana and P. hebraeus is arginine, replacing the cysteine, an important disulfide bridges element, in the position as in A. mellifera ligustica.

[Cloning and comparative analysis of the venom prepromelittin genes from four wasp species].

The total RNAs were isolated from the venom glands of female Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax and Vespa magnifica, respectively. Using the ReverAid First Strand cDNA Synthesis Kit, the first cDNAs were synthesized from the total RNA templates. PCR was carried out for 30 cycles (40s at 94 degrees C, 40s at 52 degrees C and 1 min at 72 degrees C), with a final extension of 10 min at 72 degrees C. RT-PCR products were examined by electrophoresis in 1.5% agarose gels with ethidium bromide staining. After purification using QIAquick PCR Purification Kit, the PCR products were ligated into pGEM*-T easy vector. Positive clones were identified by double digestion of Xho I and Hind III and PCR amplification and then were sequenced. The sequencing results showed that the amplified cDNAs containing the open reading frames of prepromelittin, and their lengths were all 213 bp. The ORFs were potential to encode polypeptides of 70 amino acid residues with predicted molecular weight of 7.7 kDa, including a signal peptide of 21 residues and a promelittin of 49 residues. Comparative analysis showed that the prepromelittins from different wasp species shared more than 93% identities in nucleotide sequences and more than 95% identities in amino acid sequences with each other, respectively. The sequences of prepromelittins of Polistes hebraeus, Vespula maculifrons, Vespa velutina nigrithorax and Vespa magnifica share 94%, 100%, 94%, 93% homologies in nucleotide sequences, and 95.8%, 100%, 97.2% and 97.2% identities in amino acid sequences with those of the European honey bee, Apis mellifera, respectively. There are 67 common amino acid residues among the prepromelittins of the four wasps and two honey bee species, Apis mellifera and Apis cerana. Phylogenetic relationships based on the prepromelitin nucleotide sequences were also examined using the software DNAStar5.01. The result showed that a closer relationship lied in between Polistes hebraeus, Vespa velutina nigrithorax and Vespa magnifica, while Vespula maculifrons had a closer relationship with the honey bees, Apis mellifera and Apis cerana. In conclusion, the prepromelittins were very conserved in the primary structure and the wasp insects also contain the melittins in their venoms, which are very similar to that of honey bee, although they belong to different superfamilies.