Cloning And Analysis Of AP-4 And Bmfez1 From Bombyx Mori

The study on Drosophila proves that the important development progress such as the formation of axis and the start of early embryonic development mainly depend on the mRNA from matrix. The early embryonic development of silkworm is peculiar as compared with fruit fly, so the research of silkworm maternal gene is significant to understand the embryonic development mechanism and will provide evidences for the genetic control of lepidoperan pests.Based on the information of silkworm ESTs, the cDNA of two genes in Bombyx mori unfertilized egg were cloned using the strategy of rapid amplification of cDNA ends. Concurrently bioinformatic methods were applied to analyze the obtained sequences and their induced amino acid sequences, including open reading frame ORF, exons and introns, physical and chemical properties, conserved domain, homologous sequences. The conclusion are as follows:Ⅰ,Molecular cloning and analysis of Bombyx mori activator protein 4 gene1. The cDNA of Bombyx mori activator protein 4 gene was cloned. It is 1621 bp in length and contains a 318 base pair of 5′UTR , a 993 base pair of protein coding region, a stop codon of TGA and a 307 base pair of 3′UTR. The open reading frame ORF locates in the region from 319 base pair to 1314 base pair of cDNA and encodes a protein of 331 amino acid residuals.2. The open reading frame ORF of Bombyx mori activator protein 4 gene locates in the region between 1430 base pair and 5674 base pair of silkworm genome Ctg022100. AP-4 gene contains 3 exons and 2 introns. The length of two introns are 2763 base pairs and 481 base pairs and the three exons locate in the region from 1 and 407, 408 and 570, 571 and 1621 of cDNA separately.3. The homologous analysis of amino acid sequences of AP-4 among Tribolium castaneum, Apis mellifera, Bombyx mori Helicoverpa armigera, Homo sapiens shows that there is a very similar region between 45 amino acid and 99 amino acid. It proves that the region is very conservative and is a typical conserved domain, so it may play an important role in the function achievement of AP-4.The deduced amino acid sequence of Bombyx mori transcription factor AP-4 gene showed 76 % identity to that of Helicoverpa armigera with a of score 449 and e value of 4e-124.4. Realtime quantitative RT-PCR showed that transcriptional activity of AP-4 gene can be detected in all development stages tested except newly-hatched larvae and 4th day of pupal stage, it expressed especially higher in 3rd and 4th instar larvae immediately after ecdysis. It is presumed that Bombyx mori AP-4 gene is likely to play an important role in a temporary and discontinuous expression pattern.Ⅱ,Molecular Cloning and Analysis of Bombyx mori fez1 gene1. The cDNA of Bombyx mori fez1 gene was cloned. It is 1823 bp in length and contains a 184 base pair of 5′UTR , a 1383 base pair of protein coding region, a stop codon of TAA,a 228 base pair of 3′UTR and a 25 base pair polyA. The open reading frame ORF locates in the region from 185 base pair to 1569 base pair of cDNA and encodes a protein of 461 amino acid residuals. 2. Bombyx mori fez1 gene contains 7 exons and 6 introns, it covers silkworm genome Scaffold000276 and Scaffold000435.3. The analysis of deduced amino acid sequence of Bombyx mori fezl gene with the structure region forecasting software ExPASy shows that there is a typical of conserved domain named fez1 which locates from 205 to 435 amino acid residual. The homologous analysis of the Fez1 protein suggests that the deduced protein of Bombyx mori fezl gene has a closest distance with Tribolium castaneum tumor suppressor 2 , identity and score are 35% and 241 separately, e value is 2e-61.4. According to the research results of Fez1 protein function from other species, it is presumed that the products of silkworm fez1 gene in unfertilized eggs is likely to involve in cell cycle regulation and play a role in the gather of cell canaliculus in the early embryo development...