Clone, Identification And Study On The Function Of Class B Scavenger Receptor Genes In Silkworm, Bombyx Mori

Silkworm is an important insect of agricultural economy, the silk industry is an important part in China's agricultural economy, and has been playing an active role in improving people's livelihood and sustainable development constantly. Researching in the law of silkworm vital movement and gene function in-depth is a significance for maintaining sustained and steady development of silk industry. As the accomplishing of silkworm genome framework map and detailed map, the genie informations of silkworm complete genome have been being annotated gradually, and providing a solid foundation conditions for studying on genomes and functions of various genes widespreadly and thoroughly. Scavenger receptors (SR) is a transmembrane glycoprotein in the cell surface, they can bind varied polyanions and transfer these polyanions to cells on degradation, performing many kinds of functions. The family of class B scavenger receptor (SR-B) is a small super-gene family of scavenger receptors family, these genes with CD36 domain can bind a variety of ligands, and participate in a series of the life metabolic activities, and plays an important role in physiological processes, such as in the formation and inhibition of cardiovascular disease including atherosclerosis, immune defense, clearing apoptotic cells, carotenoid metabolism and transport, pheromone sensory conduction and visual transduction and so on. This functional diversity evokes people's interest to study silkworm SR-B genes. Bombyx mori is a Lepidoptera model insect, researching the silkworm SR-B genes not only can resolve the functions of the silkworm SR-B genes, but also provides an important reference and theoretical study basis for relevant studies of other insects, specially for Lepidoptera insects, it is an importantance of revealing the functional diversity and mechanism of action of SR-B genes family. In this thesis, utilizing 9×genome data and microarray database. SR-B genes family of silkworm was identified and analyzed through bioinformatics. The structural features, expression patterns and tissues localizations of BmSCRBQ1 and BmSCRBQ4 genes from this family have been researched and analyzed, and the functions of these two genes were initially explored in cell level in vitro. The main results are as follows:1 Analysis in bioinformatics and expression patterns of silkworm SR-B genes familyBased on silkworm 9×whole genome database, utilizing bioinformatics methods,14 class B cavenger receptor genes of silkworm are identified, these genes distributed in at least five chromosomes, containing a total of 120 introns, one gene has 8.5 introns in average.. The number of silkworm SR-B genes is close to Drosophila melanogaster's. Anopheles gambiae's and Tribolium castaneum's, and more than the numbers of SR-B genes from Homo sapiens, Mus musculus, Caenorhabditis elegans, Danio rerio and Gallus gallus, indicating insect SR-B genes developed more copies after species differentiation.All SR-B genes of silkworm have characteristic domain of CD36, the similarities of amino acid sequences between silkworm SR-B genes are 20% to 39%, and conservative sites scattered, no continuous conserved sequences of amino acids in silkworm SR-B genes. The similarities amino acid sequences between silkworm SR-B genes and D.melanogaster's, A.gambiae's, A.mellifera's and T.castaneum's are 19% to 63%, between silkworm SR-B genes and mice's and human's are 20% to 35%.The results of phylogenetic analysis showed that insect SR-B genes divided into three groups, each group formed several subgroups respectively, SR-B genes from silkworm and other five insects formed orthologous relationship, no a insect species formed a unique grouping, indicating that each sub-group of such genes had been formed before species differentiato, and that the SR-B gene family in insect species could have relatively conservative functions.The results from silkworm EST and microarray database,analysis and RT-PCR testing showed that the SR-B genes of silkworm have different expression patterns, the exepression of most genes presented tissue specificity, there are the transcriptional expression of SR-B genes in main larval tissues of silkworm.2 Clone, sequences analysis and identification of silkworm BmSCRBQl and BmSCRBQ4 genesFull CDS length of BmSCRBQ1 and BmSCRBQ4 genes were cloned and sequenced. Sequence analysis showed that nucleotide sequences of BmSCRBQ4 is concordant with predicted sequences of silkworm databases, but there are three differenct types for the nucleotide sequences of BmSCRBQ1 in the silkworm strains and tissues:the nucleotide sequences of BmSCRBQ1 in some strains are consistent with database sequence predicted, called integrated type; the nucleotide sequences of BmSCRBQ1 in some strains show two alternative splicing, this is the integrity type and the deletion type which deleted exon 8; and the nucleotide sequences of BmSCRBQ1 in other strains exist 14or 15 bases mutation comparing with database sequences, called the point mutant type.The ORF lengths of BmSCRBQl integrated type or point mutant type is 1482bp, encoding 493 amino acids, with 10 exons and 9 introns; the ORF length of BmSCRBQ1 deletion type is 1377bp, encoding 458 amino acids, with nine exons and eight introns. The ORF length of BmSCRBQ4 gene is1371bp, encoding 456 amino acids, with 9 exons and 8 introns.Analyzing the structural domain and transmembrane domain by Smart, TMHMM2.0 and Motif Scan software revealed that the protein encoded by BmSCRBQ1 gene has two transmembrane domains, two terminal cytoplasmic region and an extracellular domain. There are nine N-linked glycosylation site, five casein kinase II phosphorylation sites,4 myristoylation site and a protein kinase C phosphorylation site in the protein encoded by BmSCRBQ1 gene. The protein domain characteristics of BmSCRBQl deletion type is consistent with BmSCRBQl's except the lack of two myristoylation site close to C-carboxyl terminal. The protein encoded by BmSCRBQ4 gene has a transmembrane domain in the C- carboxyl terminal with a C-carboxyl terminal cytoplasmic region and an extracellular domain including N-terminal area, no the N-amino terminal transmembrane domain was predicted. There are 7 N-linked glycosylation sites, seven casein kinaseⅡphosphorylation sites, five protein kinase C phosphorylation sites,4 myristoylation site and one tyrosine kinase phosphorylation site in the protein encoded by BmSCRBQ4 gene. These analyzing results showed that two genes do have CD36 domain, belonging to SR-Bgene.Similarity of amino acid sequences between BmSCRBQ1 and BmSCRBQ4 is about 30%, there are high conserved protein kinase C consensus sequence,5 conserved glycosylation sites, three conserved casein kinaseⅡphosphorylation sites and six conserved cysteine residues. Comparing with human CD36's and mouse SR-BI's and Drosophila ninaD's reveals high conserved protein kinase C consensus sequence, six conserved cysteine residues, and several conserved glycine, phenylalanine, and proline residues.3 The expression patterns of BmSCRBQl and BmSCRBQ4 genesRT-PCR results showed that BmSCRBQ1 and BmSCRBQ4 genes expressed in in unfertilized eggs and whole embryonic period, in majority of larvae period of Dazao strain and in cell lines BmE and BmN of silkworm. In silkworm strain Dazao tissues of 3-days-old 5th instar larvae, BmSCRBQ1 gene expressed in midgut, hemolymph, middle silk gland, ovary, head, integumentum, testis, fat body, posterior silk gland and in malpighian tubules; BmSCRBQ4 gene expressed in midgut, silk gland, head, integumentum, fat body, testis and in ovary, no expressions were detected in hemolymph, posterior silk gland and in malpighian tubules. The expression patterns of BmSCRBQ1 and BmSCRBQ4 genes expressing in mostly growth periods and in majority of larvae tissues hinted that two genes may act as housekeeping genes.Western blotting results showed that the positive signals of BmSCRBQ1 protein can be detected in ovary, hemolymph, posterior silk gland, middle silk gland, testis, malpighian tubules, fat body, head and in integumentum of 3-days-old 5th instar larvae of Dazao, no positive signal of target protein was detected in midgut. The size of BmSCRBQ1 protein is approximately 60kDa in each tissue (about 65kDa in). But. in tissues of 3-days-old 5th instar larvae of Dazao, the positive signals of BmSCRBQ4 protein were be detected only in the middle silk gland, fat body and in testes, no positive signals of the target proteins were not detected in other tissues. There are different in protein size between tissues, about 50kDa in the middle silk gland, approximately 55kDa in fat body, and approximately 60kDa in testis. In testis and ovary of 4 days 5th instar larvae of N4 stains, the positive signals of two proteins were detected, and BmSCRBQ1 protein has only one positive band with the size of about 60kDa; BmSCRBQ4 protein has three positive bands in each tissue, with the size of approximately 50kDa,57kDa and 70kDa respectively. In silkworm two cell lines BmE and BmN, the positive signals of two proteins were detected also, BmSCRBQl protein has one apparently positive band with the size of about 60kDa, BmSCRBQ4 protein has two positive bands in each cell, with the size of approximately 50kDa,57kDa(or 55 kDa) espectively. These results indicated that the proteins expressed by BmSCRBQ1 gene have single pattern and and stable size, but the proteins expressed by BmSCRBQ4 gene showed no uniform size and variform patterns, guessing that this may be related to the tissue specificity, degree of protein glycosylation or to the diversity of its structure and function.4 Tissue positioning of BmSCRBQl and BmSCRBQ4 proteinsBased on the expression results of BmSCRBQ1 and BmSCRBQ4 genes in the transcription level and protein level, using paraffin section and immunohistochemistry methods, tissues of 3-days-old 5th instar larvae of Dazao strain(such as testis, fat body, middle silk gland, haemocyte and ovaries)were selected to locate two protein in tissues. The results showed that BmSCRBQ1 protein were detected mainly in the spermatotheca membrane and intima of testis. in the plasma membrane of adipocyte, in both inner and outer membrane of silk gland cells of the middle silk gland, in the plasma membrane of original leukocyte and granulosa cells in haemocyte, and in the membrane area of follicle cells of ovary. However, BmSCRBQ4 protein were detected in the spermatogonia membrane and the outer membrane of testis, in the plasma membrane of adipocyte and in the outer membrane of silk gland cells of the middle silk gland, but no BmSCRBQ4 protein can be detected in haemocyte and ovary. The location results in different tissues showed that all two proteins exist in the constitutive tunicas of tissues and (or) in the plasmalemma area of cells, guessing that the functions of two proteins may be involved in lipid flow and metabolism, recognition and phagocytosis and transportation of metabolic waste and xenobiotics, and signaling transduction and so on.5 Initiatory study on the proteins of eukaryotic expression of BmSCRBQ1 and BmSCRBQ4 genes binding bacteriaFull ORF sequences of BmSCRBQl and BmSCRBQ4 genes were amplified and subcloned into the mammalian expression vector pcDNA3.1 after enzyme to construct the recombinant vector for expression in HEK293 cells, in order to study the functions of BmSCRBQ1 and BmSCRBQ4 genes in the cellular level in vitro using eukaryotic cell expression system. Transfection results have shown that the two recombinant vectors can express the target proteins in HEK293 cells and the target proteins obviously located in the cell membrane area of HEK293 cells.Optimizing conditions of transfection showed that the expression efficiency of the recombine vectors can be maintained in 20% to 30%. The HEK293 cells after transfection incubated with the FITC labeled E.coli and Staphylococcus aureus respectively, the samples were observed under a fluorescence microscope after immunofluorescence treatment, found that a small number of two bacteria were adhere to the edge of cell expressing BmSCRBQ1 and BmSCRBQ4 proteins, guessing that two proteins may be able to bind bacteria, further study on the binding action between them would be needed.