Study On Cloning And Analysis Of A Lethal-like Gene And Its Temporal-spacial Expression

Embryonic lethal abnormal visual gene (Elav) is a lethal gene in Drosophila whichinduces the nural system to develop and function abnormally. We cloned a Bm-Elav genesuccessfully through bioinformatics and biological experiment, based on the sequence ofELAV amio acid of Drosophila, and utilizing the Bombyx mori's EST database. TheBm-Elav cDNA was obtained by RT-PCR (Reverse Transcriptase) method from silkglandof the 5th instar larvae of DAZAO variety. Bm-Elav is a homologous gene of Elav, theORF (Open Read Frame) included 807 bp in length and encoded 268 amino acids, and thesequence is consistent with the predicted one. BM-ELAV protein includes 3 RRM motifs,and belongs to the RRM RNA family.Bm-Elav was cloned and combined with pET-28a (+) to build a fused expressionplasmid of Escherichia coli. Using the plasmid to transfeet E. coli BL₂₁, with IPTG toinduce, the fused protein of Bm-Elav was expressed successfully. This laid the foundationfor further study of Bm-Elav's function.The temporal-spatial expression of Bm-Elav was investigated in order to find out thegene's function. We optimized the method of semi-quantitative RT-PCR, and theninvestigated the differential expression of Bm-Elav gene with A3 as the inner control. Theresults showed that there was no remarkably different expression during the larval (3rd dayand 6th day of 5th instar larva, spinning time) and the pupal stage (3rd day and 6th day ofpupa) or in tissues of ovaries, fat bodies, midgets, silkglands and hemopoietic organs. TheBm-Elav gene was expressed in eggs of 0.5 h and 30 h after oviposition, but not in theactivated eggs. It is suggested that the expression of Bm-Elav in the activated eggs mightinduce abnormal vision and lead to death.