Screening And Preliminary Identification Of The Factors Interacting With The Complementary Sex-determination Gene (csd) Of Apis Mellifera

Honey bee have no specific sex chromosomes, their gender was determined by different combination of alleles of the complementary sex determination(csd) gene. Different alleles will develop into female, one allele or two of the same alleles will develop into male. The csd gene encode a protein contain with SR domain, belongs to SR type protein. This thesis mainly studied the proteins which interacted with csd gene.Used 36 h fertilized eggs of Apis mellifera of c DNA to construct yeast two hybrid library. The coding region of csd gene was cloned into the pGBKT7 carrier to establish the bait plasmid, converted to AH109 saccharomycetes and expressed the BD-X protein. Cloned the yeast two hybrid library into the carrier pGADT7, changed to the Y187 yeast and expressed the AD-Y protein. BD-X and AD-Y protein were cultured together, then screen the proteins which interacted with csd by the situation of activation of the report gene in yeast genome. The experiment filtrated two new Apis mellifera protein, Pabp2 and Fusilli with RNA-binding domain. The bee genome sequence analysis showed that forecast fusilli gene contain an ORF of 3468 bp, coding a protein of 1215 aa, with three RRM domains in the C-terminal; forecast pabp2 gene contain an ORF of 687 bp, coding a protein of 228 aa, with a RRM domain in the C-terminal.The experiment cloned the pabp2 gene of Apis mellifera and induced its prokaryotic expression. The result showed that the gene contained 8 exons and 7 introns, their boundary are accord with GT-AG rule; The pabp2 gene contain an ORF of 687 bp, coding a protein of 228 aa, the molecular weight of 25.73 kDa, with a RRM domain. The blast result as follows: the identity with Camponotus floridanus, Anopheles gambiae, Drosophila melanogaster, Bombyx mori, Aedes aegypti are 98%, 80%, 78%, 78%, 74% respectively. We also induced a protein Pabp2 about 25.73 kDa by prokaryotic expression.Finally we cloned Amrbp1 and Amrbp7 gene and induced their prokaryotic expression. The results showed that Amrbp1 contained three alternative splicing forms respectively named Amrbp1-R1, Amrbp1-R2, Amrbp1-R3. The ORF are 393 bp, 501 bp, 489 bp respectively. Their protein are 130 aa, 166 aa, 161 aa respectively. The molecular weight are 14.97 k Da, 19.29 kDa, 18.83 kDa respectively. Amrbp1-R1 contained 5 exons and 4 introns. Amrbp1-R2 and Amrbp1-R3 included 7 exons and 6 introns. Blast result showed that the identity with Nasonia vitripennis, Solenopsis invicta, Aedes aegypti, Bombyx mori, Drosophila melanogaster, Anopheles sinensis are 96%, 93%, 80%, 78%, 78%, 68% respectively. This study cloned two forms of Amrbp7 gene were named Amrbp7-P1 and Amrbp7-P2. The ORF are 486 bp, 501 bp respectively. The protein of 161 aa, 166 aa respectively. The molecular weight are 18.65 kDa, 19.29 kDa respectively. Amrbp7-P1 contained 5 exons and 4 introns. Amrbp7-P2 contained 6 exons and 5 introns. Blast result showed that the identity with Nasonia vitripennis, Solenopsis invicta, Bombyx mori, Drosophila melanogaster, Aedes aegypti, Anopheles sinensis are 89%, 81%, 71%, 67%, 67%, 54% respectively. Amrbp1 and Amrbp7 are contained a RRM and a SR domain. We also induced a protein RBP1 about 20 kDa by prokaryotic expression.