Cloning, Identification And Functional Analysis Of The Apoptosis-related Gene BmDronc And BmBuffy In Bombyx Mori

So far, there are mainly two primary programmed cell death signaling pathways, apoptosis and autophagy. Apoptosis as well as cell proliferation and cell differentiation plays a crucial role in many biological processes, such as the growth and development, organ formation, body shape, the maintenance of internal homeostasis. The imbalance of apoptosis can lead to tumorigenesis, autoimmune disorder, nerve degeneration, the lack of hematopoietic stem cells, directed infertility and so on. The mechanisms of apoptosis are being clarified in model organisms especially in nematodes. fruit flies and humans. Compared to flies and human beings, the apoptosis networks in neroatodes is much simpler, while is most complicated in mammal.Silkworm is complete metamorphosis of insects, through the egg. larva, pupa and adult stages of four stage to complete its life cycle. In addition, because of the silkworm's rearing easily, short generation, more offsprings, clear genetic background, high economic value and other advantages, International Association of Invertebrates recognized silkworm as the insect mode of Lepidoptera in 2002. Investigation into apoptosis in Lepidotera began at the same time as Drosophila from last 1960s. Apoptosis research in silkworm has been lagged until the 1990s. Now, apoptosis research in silkworms mainly focus on cloning and verification of apoptosis-related genes, which is incongruity with the model insect state. At the same time, the apoptotic mechanism can't be completely replaced by other species. Therefore, the studys on apoptosis in silkworm have to be carried out immidiently.The apoptosis-related gene and the expression patterns were analyzed systematically using silkworm genome data, ESTs data and microarray data by the bioinformatic method. At the same time, the new genes were identified by cloning and sequencing. On this basis, the apoptotic pathways that may be exist in silkworm were speculated. Furthermore, the functional study on the apoptotic genes BmDronc and BmBuffy were performed by RT-PCR. prokaryotic expression, immunofluorescence and ectopic expression and so on. The main results obtained are as follows:1. The bioinformatics analysis and veritification of the apoptosis-related genes in silkworm.Using the assembled 9X coverage genome sequence,52 putative silkworm apoptsis-related genes were identified by bioinformatic methods, including five members of the caspase family, one member of the Bcl-2 family, two members of the TNF superfamily (TNFSF), and four members of the baculovirus IAP repeat (BIR) domain family in Bombyx mori. Seventeen genes have been previously included in NCBI, other thirty five apoptosis genes were identified newly. Forty apoptotic genes matched at least one expressed sequence tag (EST), and the remaining genes had incomplete ESTs or none ESTs. To veritify the apoptosis genes,32 genes primers were designed according to their predicted DNA sequences and the RT-PCR were carried out using the cDNA isolated from the different development stages of silkworm and BmE cells exposed to different stressors. As a result. Twenty three genes were cloned and sequenced successfully; five genes were detected by RT-PCR but not sequenced successfully and four genes were not detected. The results of RT-PCR show that most of key apoptosis-genes are expressed in Bombyx mori.The comparing key apoptosis-related gene numbers in various species showed that there are much more homologous genes in insects. However, many genes homologues were not hit in our silkworm databases, including almost all genes of the Bcl-2 and TNFSF families, and caspase-6/-7, Hid, Grim, and Sickle of the RHG family.Compared with the genes in the mammal apoptotic pathways, the apoptosis-related factors identified in silkworms covered almost the critical junctions in the apoptosis pathways. In all. the intrinsic and extrinsic pathways described in other models maybe potentially exist in Bombyx mori.2. Cloning and functional analysis of the apoptosis-related genes BmDronc in silkworm.The Caspase-9 homolog BmDronc cDNA including the complete ORF sequence was cloned. The results analyzed indicated that BmDronc located on the No.10 chromosome, having two splicings:BmDroncL and BmDroncS. The ORF length are 1248bp and 552bp, coding 415aa and 183aa, the molecule weight are 47.76kDa and 21.39kDa, the isoelectric point are 7.65 and 9.35, respectively. The similarity and identify of BmDronc with other species are 40%-53%and 23%-33%, respectively. The BmDroncL has three classic domains:CARD, large subunit and small subunit. While the large subunit is lacked in the BmDroncS. The protein sequences of the Caspase-9 homologues in different species were aligned to construct the phylogenetic tree, as showed that Caspase-9 is highly conserved from the lower animals to higher animals.To detect the expression of BmDronc, real time PCR was carried out using the cDNA from the different developmental metamorphosis stages tissues and whole body. And the results indicated that BmDronc plays a crucial role in the PCD during the metamorphosis of silkworm.The ectopic expression of BmDronc showed that the typical apoptotic features were present in the sf9 cells transfected with A4-BmDronc-DsRed, such as cell concentration, nuclear shrinkage, and even apoptotic boby. All of these evidences suggested that BmDronc can promote the apoptotic program in Sf-9 cells.3. Cloning and functional analysis of the apoptosis-related genes BmBuffy in silkworm.The complete 1632bp cDNA of Bcl-2 family member BmBuffy was obtained by the 5'and 3'RACE PCR. The 879bp ORF code 292aa,containing BH1, BH2, BH3 domain and a transmembrane region. The phylogenetic tree of BmBuffy with the Bcl-2 family homologues showed that BmBuffy is more similar to Buffy of Apis mellifera and Drosophila, and clustered with the mammal Bcl-2 family member Bok in one class. The similarity of BmBuffy with Drosophila and human homologues are 49% and 51% , respectively. The traditional genetic linkage analysis revealed that nscaf2983 and nscaf3077 locate on the No.4 chromosome. Although the position of the nscaf2981is uncertainly, it can be conclued safely that Bmbuffy locate on chromosome No.4 according to the location of nscaf2983 and nscaf3077 and the position of the nscaf2981, which is in the No.7 chromosome end.To detect the expression of BmBuffy, real time PCR was carried out using the cDNA from the tissues and whole body during different developmental and metamorphosis stages. The results revealed that the expression of BmBuffy in the different tissues and whole body don't changes regularly, and be inconsistent between the two key point during the metamorphsis in silkworm. So it is considered that the BmBuffy is likely to have other physiological functions besides apoptosis.The BmBuffy_P_pET21 a recombinant (BmBuffy_P lacking the transmember region) was constructed by the recombination technology in vitro. Then prokaryotic expression, BmBuffy_P protein purified, affinity chromatography and electro eluter, immunization in vitro, antibody purification were carried out. Finally, BmBuffy_P polyclonal antibodies were obtained. Immunofluorescence experiments were carried out using the BmE cells in silkworm labled with endoplasmic reticulum and mitochondrial probes. The results showed that BmBuffy is not located on the endoplasmic reticulum. while located on the mitochondria. These results illustrated that BmBuffy may be possibly involved in the regulation of mitochondrial apoptotic pathway.The BmBuffy_pEGFP and BmBuffy_P_pEGFP were expressed in the HEK293 cells expression system. The results revealed that there was any changes among the cells transfected with BmBuffy_pEGFP, BmBuffy_P_pEGFP and pEGFP-N1, respectively, as showed that the apoptosis program didn't happened. So it is speculated that if BmBuffy play roles during the regulation of apoptosis, it maybe regulated at the upstream of the BmDronc.