Isolation and expression profiling of fruitless gene in Anopheles stephensi / 中华实验和临床病毒学杂志

Objective@#To isolate, identify and analyze the sex-determining gene fruitless(Anstfru)of malaria-transmitting mosquito Anopheles stephensi.@*Methods@#The full length cDNA of the gene Anstfru was obtained by using bioinformatic and molecular biological method . RT-PCRmethod was used to validate the sex variable splicing pattern and expression time characteristics. The structural features and molecular evolutional features of FRU protein of Anopheles stephensi were analyzed via comparison with FRU protein of known species.@*Results@#The full length of Anstfru gene was isolated and identified, and sex-specific mRNA of the gene could form in female and male mosquitos through variable splicing. The Anstfru began to be expressed from early 1st-2nd stage larvae embryo, the quantity of expression increased subsequently and displayed the highest expression level in adult stage. The FRU protein had the sequence-conservative BTB and zinc finger functional domains.@*Conclusions@#Anstfru gene showed conservative functional domains and sex-determining gene fru expression features in mosquito and further in-depth studies on which will facilitate the application of techniques separating female mosquitos from male mosquitoes, and sterile insect technique (SIT)/technology in prevention and treatment of mosquito-borne infectious diseases.

Identification of MicroRNA-like Viral Small RNAs from AaeDV / 病毒学报

Several virus families have been shown to encode microRNAs (miRNAs), which have roles in the infection and replication of viruses in host cells. These virus-encoded miRNAs are identified in double-stranded DNA virus (dsDNA virus) and in several RNA virus families, but not in single-stranded DNA virus (ssDNA virus). We used a bioinformatics approach based on VMir, miRNAFold and MaturePred software to predict virus-encoded miRNA-like small RNAs from the genome of a ssDNA virus: Aedes aegypti densovirus (AaeDV). Northern blotting and stem-loop reverse transcription-polymerase chain reaction (RT-PCR) were used to detect predicted small RNAs. A miRNA-like small RNA termed "AaeDVMD" was identified by stem-loop RT-PCR from predicted candidates. This is the first report demonstrating that a ssDNA virus can encode miRNA-like small RNAs. These data will aid further exploration of the interaction between the AaeDV and its mosquito host.

Isolation and expression profiling of transformer 2 gene in Aedes aegypti / 南方医科大学学报

<p><b>OBJECTIVE</b>To isolate, identify and analyze the sex-determining gene Transformer 2 (Aaetra2) of the major vector mosquito Aedes aegypti.</p><p><b>METHODS</b>tBLASTn program, RT-PCR and RACE methods were used to obtain the full-length cDNA of Aaetra2. Multiple alignments of nucleotide and amino acid sequences were conducted, and the different domains in tra2 protein were indentified. RT-PCR of the total RNA extracted from different tissue from the mosquitoes in different developmental stages was performed using specific primers.</p><p><b>RESULTS</b>Two genes, namely Aaetra2-α and Aaetra2-β, were identified in different supercontig locations. The multi-transcripts were expressed by means of alternative promoters or terminators. The different domains in tra2 protein were defined as RS-rich N-terminal region, RNA recognition motif-RRM, linker region, and RS-rich C-terminal region. Both Aaetra2-α and Aaetra2-β showed sustained expression throughout the developmental stages of Ae.aegypti, and in all the tissues without a sex specificity.</p><p><b>CONCLUSION</b>Aaetra2 gene has multiple isoforms and is mapped to multiple locations in the genome. Aaetra2 has conservative functional domains of the sex-determining gene tra2. For Ae.agypti, Aaetra2 shows the potential as a new target for release of insects carrying a dominant lethal (RIDL) technology based on transgenic mosquitoes.</p>

Construction of a Yeast Model for Screening Aedes albopictus Ecdysone Agonist Pesticides / 中国寄生虫学与寄生虫病杂志

Objective To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. Methods The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene——green fluorescence protein(GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site(pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcrip-tion level of GFP in the tebufenozide affected yeast and the control. Results In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tubufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tubufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. Conclusion The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

Improved Preparation of Pure Alive Eggs of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

To prepare a large amount of pure alive Schistosoma japonicum eggs, rabbit was infected with 2000 cercariae and its liver was taken aseptically 38-45 days after infection and homogenized. The homogenate was screened through different sieves(60, 120, 200, 300, 360 meshes per inch respectively), and washed with 1.2% NaCl. The eggs and leftover were then digested with 0.25% trypsin for 2 hours, sieved over 360 meshes per inch and washed with RPMI 1640 medium. The collected eggs reached to (95.1?6.4)% of live eggs, with a high efficiency.