Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

[Cloning and analysis of adenine nucleotide translocase gene in Helicoverpa armigera].

A cDNA clone encoding the ADP/ATP translocase in Helicoverpa armigera has been identified by RT-PCR, 5'and 3'RACE methods. Sequence analysis shows that it is 1,190 bp long and contains a single open reading frame (ORF, 133-1,033 bp) encoding a protein of 300 amino acids (GenBank submission number, AY253868). The protein has a 22 aa signal peptide on its N-terminal, which leads the protein locating onto the inner membrane of the mitochondria. It also has three conserved domains of the mitochondrial carrier protein forming a channel to exchange ATP and ADP energy molecule through the inner membrane of the mitochondria. It shows extensive similarities to the known ADP/ATP translocase poly-peptides. The ADP/ATP translocase similarity was up to 90% in the Lepidoptera.

[Agrobacterium tumefaciens-mediated transformation of the pathogen of Botrytis cinerea].

Employing gfp as a reporter gene and hygromycin gene (hph) as a selection marker, the recombinant vector pKPG was constructed and transformed into fresh conidia of Botrytis cinerea via Agrobacterium tumefaciens. Transformants were identified by PCR analysis of gfp and hph cassette, green fluorescence observation with microscope and Southern hybridization. Results confirmed that target genes were successfully integrated into the genome of Botrytis cinerea.

[Cloning and mapping of DpKettin gene on Z chromosome of the pine caterpillar by real-time quantitative PCR].

The ratio of Z chromosome number to autosomal chromosome (A) is different between male and female lepidoptera insects. In males: 2Z : 2A=1, and in females: Z : 2A=0.5. The ratio of the copies of gene (such as K) on Z chromosome number to the copies of gene (such as N) on autosomal chromosome (K/N) is also different between male and female. In males: 2K : 2N =1, and in females: K : 2N=0.5. The DpKettin gene of the pine caterpillar Dendrolimus punctatus was cloned with the primers designed according to the sequences of the BmKettin of the silkworm and the HaKettin of the cotton bollworm. The ratio of copies between DpKettin and ANT (adenine nucleotide translocator) in male and female was determined by the real-time quantitative PCR technique. They were 1.0 in males and 0.5 in females. These ratios were equal to the ratios of the copies of gene on Z chromosome number to the copies of gene on autosomal chromosome. It indicates that DpKettin is located on Z chromosome in the genome of the pine caterpillar.

[Cloning and structural analysis of MLP in the silkworm, Bombyx mori].

The LIM domain is found in a wide variety of eukaryotic proteins that regulate gene expression and cell differentiation during development. Muscle LIM protein (MLP) gene in Bombyx mori has been cloned by blasting its EST database and PCR test in present report. The resulting sequence covers 2 327 bp of cDNA (GenBank accession No. DQ311195). It has a complete open reading fragment and encodes a 494 amino acid protein. Genomic DNA sequence contains 11 exons and 10 introns, with intron splicing following the GT-AG rule. M.W. and PI of the predicted MLP in Bombyx mori are 53.03 kDa and 8.29 respectively. A single LIM domain linked to a glyscine-rich region is found in a previously deposited LIM protein (AAR23823) in Bombyx mori. MLP identified in this report encodes a protein with five tandem LIM-glycine modules. The two LIM proteins could be produced by alternative splicing and both are probably involved in muscle cell differentiation. This work provides foundation for further research on the in vivo function of MLP.

[The antibacterial effect of cecropin B on pseudomonas aeruginosa infection of wounds in mice].

OBJECTIVE: To investigate the antibacterial effect of a particular antimicrobial peptide Cecropin B(CB) on Pseudomonas aeruginosa infection of wound in mice. METHODS: Thirty ICR mice were enrolled in the study, and the Pseudomonas aeruginosa infection model was reproduced by excision of the full layer of dorsal skin with an area of 1 cm x 1 cm. Then they were randomly divided into C ( control, n = 10, with wet compress of isotonic saline at 3 postinjury hour( PIH) ) , M (with hydropathic compress of 100 g/L mafenide at 3 PIH), A (with wet compress of 1 000 mg/L Cecropin B at 3 PIH) groups. The changes in body temperature and hemogram in each group were determined before and 4 days after injury. Quantitative examination were used to detect the quantity of bacteria in muscular tissue of the wounds, and the survival of the mice were observed on 4 post-injury day( PID). RESULTS: The wounds were moist with more exudation in C group,while that in other groups were dry without obvious exudation. The body temperature of the majority of the mice in each group were elevated, but the number of leucocytes in each group was lowered after operation. The quantity of bacteria in muscle in A group[ (42 +/- 50) CFU/g] was obviously lower than that in M group [(886+/-804) CFU/g, P <0.05] , and it was all obviously lower than that in C group[ (41 +/-28) x 10(5) CFU/g, P <0.01]. The number of surviving mice after 4 PID in C group was evidently smaller than that in A and M groups( P <0. 05). CONCLUSION: The cecropin B possesses obvious anti-bacterial effect on the Pseudomonas Aeruginosa infected wounds of ICR mice, and it can reduce the mortality.

Cloning and alternative splicing analysis of Bombyx mori transformer-2 gene using silkworm EST database.

We have identified Bombyx mori transformer-2 gene (Bmtra-2) cDNA by blasting the EST database of B. mori. It was expressed in the whole life of the male and female silkworm and was observed as a band of 1.3 kb by Northern blot analysis. By comparing corresponding ESTs to the Bmtra-2 DNA sequence, it was revealed that there were eight exons and seven introns, and all splice sites of exons/introns conformed to the GT/AG rule. Bmtra-2 pre-mRNA can produce multiple mRNAs encoding six distinct isoforms of BmTRA-2 protein using an alternative splicing pathway during processing. Six types of Bmtra-2 cDNA clones were identified by reverse transcription-polymerase chain reaction. All isoforms of BmTRA-2 protein contain two arginine/serine-rich domains and one RNA recognition motif, showing striking organizational similarity to Drosophila TRA-2 proteins.

[Cloning and expression of the cecropin B-thanatin hybrid antimicrobial peptide in Escherichia coli].

A 44-residue hybrid peptide (CB (1-24)-Arg-Ser-Tyr-Tan (4-21)) incorporating 1-24 residues of cecropin B (CB) and 4-21 residues of thanatin (Tan) was designed and constructed. The CB-Tan gene was cloned into expression plasmid pGEX-3X and expressed in E. coli BL21. The fusion protein was purified by affinity chromatography. After digested with enterokinase the gene product released with antibacterial activity and gave one band in Tricine-SDS-PAGE.