| In recent years, the bioinformatics has becoming one of the focuses in Genetics and Biology. In silico cloning the novel genes based on the EST database is an important aspect in the field. The honeybee (Apis melliferd) is important in the agricultural community as a producer of honey and as a facilitator of pollination. It is a model organism for studying human health. In addition, biologists are interested in the honeybee's social instincts and behavioural traits. However, the genes of honeybee previously reported were few. After the Honeybee Bee Genome Project (HBGP) was completed, using EST data to clone the novel genes in honeybee possesses theoretical significance and practical value.In this thesis, we used the ESTBlast to in silico clone novel honeybee genes. Moreover, we predicted the function of the putative peptides encoded by the genes. The aim of this study is to establish available cloning gene approach based the high similarity of homologous gene sequences in different species.1 cloning and analysis of honeybee ant geneA novel honeybee gene (ant) full-length cDNA of 1 251bp (GenBank Accession, AY332626) deduced encoding adenine nucleotide translocator was cloned by Blasting the honeybee EST database with the fruit fly homologous ant gene cDNA sequence information as a querying probe.Searching the cDNA sequence for potential coding regions by ORF finder (NCBI), an entire open reading frame (ORF) of 300 amino acids was detected with a potential start -codon at the 29th site and a stop codon at the 931st site of the sequence. The results of the multiple sequence alignment revealed the putative peptide encoded by honeybee ant gene is complete, and ant is actual functional gene in honeybee. To examine the accuracy of in silica cloning cDNA sequence, we cloned by RT-PCR and sequenced a cDNA fragment covering the coding region of ant gene. The result confirmed that the in silico cloning cDNA sequence was very accurate.The multiple sequence alignment showed the ant cDNA sequence of honeybee shares 76%, 72%, 71%, 70%, and 75% identity with the ant cDNA of Drosophila117melanogaster, Bombyx mori, Manduca Sexta, Anopheles gambiae, and Lucilia cuprina, respectively.The full-length genomic sequence of 5 774bp of honeybee ant gene (GenBank Accession, AY568009) was cloned and sequenced. The result of analysing the sequence using the Gene Finder software showed there are 3 introns at the position of 1 717th~3 078th site, 3 341st~3 442nd site, and 3 678th~3 760th site, respectively; and 4 exons with length of 192bp, 261bp, and 562bp, respectively. 2 potential promoter sequences of 1 552bp honeybee ant genomic sequence in front of the start codon of the ORF were predicted at the position of 1 076th~l 126th site (score=0.87) and 1 242nd~l 292nd (score=0.96).The honeybee ant encoded a putative peptide of 300 aa with a Mr of 32.987kDa, and theoretical pl=9.1\. There are 3 hydrophobic regions at the position of ll^-lSe* site, 174th~196th site, and 211st~233th site of the deduced aa sequence, respectively. Prediction of TMHMM-2.0 suggested 3 transmembrane regions at the position of 114^-136* site, 174th-!96th site, and 211st~233th site of the deduced aa sequence, respectively. SignalP-2.0 predicted the signal peptide at the position of 22nd site.Blasting the aa sequence against the protein database in NCBI GenBank showed very high similarity to a plenty of adenine nucleotide translocators (ANT) from other species. And blasting the aa sequence against the conserved domain database suggested 3 regions of similarity to the conserved domain of 'Mito_carr (Mitochondrial carrier protein)' at the position of 9癕07th site, 114-210th site, and 211st~300th site, respectively.The deduced aa sequence of honeybee ant was aligned against related ANT sequences of other species such as Drosophila melanogaster, Bombyx mori, Manduca Sexta, Anopheles gambiae, and Lucilia cuprina, the multiple sequence alignment showed the identity of 81%, 81%, 83%, 75%, and 82%, respectively, and the similarity of 86%, 89%, 90%, 83%, an...
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