Clonging, Expression Profile And Function Of Abdominal-A Gene In Silkworm, Bombyx Mori

Abd-A,which was first reported in Drosophila melanogaster,is one of the fateful genes during the development of insects.Abd-A participate in regulation many vital processes in D. melanogaster,such as the formation of abdominal structure,differentiation of anterior-posterior (AP)body axis,migration of germ cells,gonadial development,cardiac differentiation, development of nervous system,formation of midgut and development of fat body.Abd-A is widely conserved in many species.Since its first report in D.melanogaster,abd-A homologues have been identified in many other insects,Schistosoma mansoni and mammalian. Silkworm is the model organism of Lepidoptera,and the study of silkworm embryinic development is of great important.Until recently,two mRNA splicing patterns of abd-A gene have been reported in the silkworm,but study on its function is not yet reported,we cloned the silkworm abd-A homologue,detected its expression of different stages during silkworm development and preliminarily studied on its function by RNA interference technique.The main reserch contents and results are as follows.1.Clonging and character of silkworm abd-A geneDepending on the results of bioinformatics analysis,we designed primers for silkworm abd-A gene and then cloned this gene.The cloned mRNA length of the abd-A gene comprised an open reading frame and part sequence of 3' UTR.Results showed that silkworm abd-A gene had at least three splicing pattern of mRNA.Each length is 1032 bp,1044 bp and 1059 bp,respectively coding for 343 amino acids,347 amino acids and 352 amino acids.During the three patterns,first and second pattern have been reported on NCBI,and the third one is the new result from the present study.All of them contain 3 extrons,and all the extron/intron splicing boundaries obey the CT/AC rule.The difference between the three splicing pattern is the size of second extron which length is 33 bp,45 bp,60 bp,respectively.Protein sequence alignment shown silkworm ABD-A protein respectively share 67%and 57%identity with the ABD-A of T.castaneum and Drosophila. 2.Expression profile analysis of silkworm abd-A gene mRNAFirst,we detected the expression profile of abd-A gene during silkworm embryogenesis. Results of RT-PCR showed the expression of abd-A gene started around 22 h after egg laying in silkworm embryo,then increased gradually,and decreased in newly hatched larva.This expression pattern is similar to that of Drosophila abd-A homolog during embryogenesis.We deduced that the silkworm abd-A plays important roles in formation of germ band and differentiation of abdominal and midgut in silkworm.Moreover,abd-A gene is expressed in the unfertilized egg.It could be deduced that abd-A gene plays important roles in oogenensis of silkworm.And then,RT-PCR detection revealed that abd-A gene expressed in all detected tissues from silkworm 5th instar larva,such as head,epidermis,ovary,tetise,malpighian tube,silk gland, hemolymph,fat body,and midgut.The result showed that abd-A gene participates developmental regulation of many tissues in silkworm.3.RNAi of silkworm abd-A gene during silkworm embryogenesisUsing normal N4 silkworm eggs as injection materials,RNAi experiment on abd-A gene was performed.Results showed that there were mainly two mutant phenotype,typeⅠand typeⅡ.The appearance of typeⅠmutations was that abdominal appendages in 3^th～6^thabdominal segment were totally absent,while only part of abdominal appendages absent in 3^th～6^thabdominal segment in typeⅡmutations.The result revealed that abd-A gene was essential for determinative decisions druing the development of abdominal appendage in 3^th～6^thabdominal segment.Moreover,the effect of RNA probe concentration on mutant phenotype was performed.Results showed that the more probe dose of inject,the higher ratio ofⅠtype mutations and total ratio of variation, otherwise,the less dose of probe,the lower ratio ofⅠtype mutations and total ratio of variation. The result revealed that the appearance of mutations was dose-dependent,so it could be presumed that occurrence of different mutant phenotype was caused by the expression of abd-A gene which was repress at different degree.