Studies On The Expression And Regulation Of BmApt In The Organ Development Of Silkworm

Transcription factor Apontic(Apt)is a bZIP protein and it plays a key role in the development process of the tissues of Drosophila embryo,such as the head,wing,heart,trachea,eye,nervous system and so on.Apt homologous protein of human,Fibrinogen silencer-binding protein(FSBP),is a cancer related factor.It can also affect the development of the lung,brain,heart and other tissues of human.In the silkworm,it is known that Bmapt had an effect on the color diversity by regulating the synthesis of the epidermal pigment in the nature.However,the functional research of Bmapt is not clear in other tissues.Bombyx mori is a lepidoptera model species which has many adventages,such as a single diet structure,short feeding period,being easy to dissection,richer genetic resources and a longer history research.Compared with fruit flies,silkworm is easy to experiment becase of a simpler structure and a larger body.In addition,with the completion of genome sequencing of silkworm,it will make a great progress on the research of the gene function of silkworm.Therefore,it has a great development prospect to study the function of genes with silkworm as experimental material.In this experiment,we adopt the silkworm as the research object and analyzed the structure of BmApt protein and the relationship of the evolution of silkworm.Then we employed the immunostaining and RNA interference technology to study the expression patterns of Bmapt in the nervous system and silk gland of silkworm and the regulating relationship of BmApt with Netrin and Frazzled that are a kind of axons guiding factor.The main results are as follows,(1)Protein structure analysis of BmAptAn alignment was performed on the basis of protein sequences of BmApt,DmApt and HsFSBP by using DNASIS MAX Version 3.0 software.The results showed that most of sequences of Myc/SANT motif and basic DNA binding domain of BmApt,DmApt and HsFSBP were consistent and it illustrated that they are very conservative in their Myc/SANT motif and basic DNA binding domain.However,for the distance between Leucine zipperdomain and basic DNA binding domain,BmApt of silkworm is far than DmApt of fruit fly and it may be associated with the combining ability of DNA binding domain.(2)Evolution analysis of BmAptPhylogenetic tree was constructed on the basis of protein sequence of BmApt of silkworm and the homologous of Apt of other species from NCBI database by using MEGA 5.05 software.The results showed that the protein sequence of BmApt was tightly related with PaApt of Pachliopta aristolochiae of lepidoptera insect,but had a farther evolutionary distance with Apt of the hymenoptera and diptera.(3)Preparation of BmApt antibodyAccording to the CDS full-length sequence of Bmapt gene in NCBI database,the primers were designed and the target gene Bmapt was obtained by PCR.Recombinant expression vector pET28a-Bmapt was constructed by the plasmid and then expressed in BL21 competent cells to get fusion protein.The results was obtained by SDS-PAGE electrophoresis and showed that the fusion protein existed in cells in the form of inclusion body.In addition,after immunizing the rabbits with the purified fusion protein,we extracted and purified the serum to prepared as polyclonal antibody.And we detected antibody titer by ELISA and Western blot.The results showed that the antigen and the antibody of BmApt can produce specific binding reactions.(4)Expression pattern analysis of BmApt in different tissuesWe immunostained the nervous system and silk gland of different developmental periods and the brain,trachea and fat body of day 3 of the 5th instar larva by BmApt antibody.The results are as follows.In nervous system of silkworm,the expression of BmApt in central nervous at stage 20,21,22 of the embryo and the brain of the larvae,which indicated that BmApt was associated with the development of nervous system of silkworm.In the trachea and fat body,BmApt can be expressed in their cell nucleus,indicating that BmApt had an impact on the development of trachea and fat body of silkworm.For different periods of silk gland,BmApt and Cyclin E were colocalized in the cells of silk gland.These results revealed BmApt may influence the development of silk gland of silkworm by regulating cyclin E.(5)siRNA interference analysis of BmaptThe function of Bmapt gene was studied with siRNA induced RNAi technology.Wedesigned and synthesized two Bmapt siRNA fragments that were mixed and diluted into a50μM system and then were injected into the early embryos of silkworm.The control embryos were injected with universal primer NC siRNA(NC).The results showed that the expression of Bmapt in the nerve cells of silkworm embryos injected with Bmapt siRNA was significantly less than that of embryos injected with NC siRNA.In the Bmapt RNAi embryos,the distance that bilateral axons to the midline is wider compared with that of the control.These data indicated that Bmapt may have an attractive function to the axon guidance of the silkworm.To further verification,we carried out the fluorescence quantitative experiment.The results showed that the relative expression of BmnetrinA,BmnetrinB and Bmfrazzled were significantly reduced and that indicated Bmapt controls the development of nervous system by activating the expression of BmnetrinA,BmnetrinB and Bmfrazzled.