Molecular Characterization Of Vitellogenin Gene And The Expression In Honey Bee, Apis Cerana Cerana

Chinese bee(Apis cerana cerana) is one of Eastern bee subspecies, also the native bee resources in China. It has excellent apicultural properties and irreplaceable economic, social and ecological roles. It occupies a pivotal position in apicultural development of our country.In the future, the protection and exploitation of the special resources need for further systemic researches urgently. Presently, research results for Chinese bee mainly focused on the morphological geography and other aspects of systematology, for a particular functional gene research is still in its infancy. Vitellogenin aspact is one of the most active fields on physiological and biochemical researches of bees nowadays. In this thesis, DNA cloning,sequencing and bioinformatics methods were used to study the full DNA sequence and the biological information of Chinese bee vitellogenin gene; SSCP-PCR techniques for exon genotypic characteristics of vitellogenin gene; qRT-PCR techniques for vitellogenin mRNA transcripts of Chinese bee queens, workers and drones in their adult days; SDS-PAGE and Western blot tests were done for vitellogenin synthesis of Chinese bee three castes in their adult stages. The main findings are as follows:1. The primary structure of vitellogenin gene DNA in A.c.ceranaThe vitellogenin DNA sequence contains 6496 bp, wherein C and G content of 46.14%.Compared with Italian bee(A.mellifera ligustica), the homology is 92%. The whole DNA sequence constructs seven exons, encoding a total of 1689 amino acids.2. Vitellogenin protein structure prediction of A.c.ceranaThe amino acid sequence of vitellogenin contains a recognition factor site, this site is a serine-rich region, a typical structure of eukaryotic gene, has a zinc finger structure at846~856 site that HSFPTETgLPF, has 89 post-translational modification sites, among them75 phosphorylation sites are included. This indicates that phosphorylation is the most important post-translational modification way of vitellogenin in A.c.cerana.3. Genotype of vitellogenin gene exon in A.c.ceranaAllele A, B, L, M, N and genotype AA, AB, LM, LN were detected in the fifth exon of vitellogenin gene, Hainan Chinese bee, wherein genotype AB and LN were specific and dominant. Neither AB nor LN was found in the other bee samples. The Chinese bee population in Hainan was in the situation of Hardy-Weinberg balance according to the SSCP data, with independent genetic characteristics. I conclude that the length indicators, proboscislength, wing length, wing width and the length of tergum3 + 4, etc., are associated with the unique genotype AB and LN.4. The transcription pattern of Vg mRNA in Chinese bee adult workersThe expression pattern of vitellogenin gene, in adult A.c.cerana worker bees, is different from A.mellifera, exhibiting intermittent transcription. On day 4, day 12 and day 19 after emergence, mRNA transcription peak values occur. On day 1, day 8, and day 17, the activity of mRNA transcription comes to a trough.In head, thorax and abdomen vitellogenin can be detected, where the expression abundance of head lowest, followed by thorax, abdomen has the highest mRNA levels(P<0.05). Regardless of the head, thorax, abdomen or hemolymph, vitellogenin expression abundance in worker bees, A.c.cerana is significantly lower than that of Italian bees(P<0.05).5. Vitellogenin gene expression in queen bees, A.c.ceranaWithin a week after emergence, the vitellogenin abundance increases slowly. After 6 days,sexual maturity, the expression level of vitellogenin increases rapidly. During oviposit period,vitellogenin protein has the highest expression level.Similar to worker bees, vitellogenin gene can be expressed in the queen’s head, thorax and abdomen. Vitellogenin protein in abdomen is the most abundant(P<0.05).6. Vitellogenin gene transcription and protein synthesis in drone bees, A.c.ceranaVitellogenin mRNA can only be detected in drone bees on day 1-3 after emergence, but with weak signals. After the fourth day, mRNA signals can no longer be detected. Vitellogenin protein signal were not detected to be present from all the drone hemolymph samples either.Presumably, drone bees have trouble vitellogenin synthesis, resulting in the negligible protein quantity. The function of vitellogenin protein may be degraded in drone bees.All the results of this study will be the theoretical basis for the development and utilization of functional genomics in honey bees, will make a difference on the protection for the special bee resources in China.