Study On The Physiological Function And Immunity Of The Transgenic Drosophila Melanogaster Expressing A Single Whey Acidic Protein Domain-containing Peptide Of Chinese Shrimp(Fenneropenaeus Chinensis)

The single Whey Acidic Protein domain-containing peptide(WAP)from Chinese shrimp(Fenneropenaeus chinensis)has dual functions including broad-spectrum antimicrobial activities and proteinase inhibitory activities.Therefore,it has the potential of developing antibiotic alternatives.Our previous studies have found that the products prepared with UAS-wap/Gal4 transgenic Drosophila melanogaster constructed using the wap gene showed not only higher antibacterial activities and protease inhibitory activities than that of WAP recombinant proteins expressed by Escherichia coli or yeast,but also better antibacterial and anti-inflammatory effects than that of antibiotics in animal experiments.But,it is still unclear for the molecuLar mechanism of the UAS-wap/Gal4 flies with high antibacterial activity.Based on this,this thesis has conducted two aspects of studies.On the one hand,the physiological functions of UAS-wap/Gal4 transgenic D.melanogaster were studied.Its main contents included two parts.The first part was an observation on the growth cycle,shape,weight of UAS-wap/Gal4 transgenic D.melanogaster and its control flies respectively in normal condition and under infection of different pathogen.The second one was an observation on the spawning,pupation rate,hatching rate and life span of UAS-wap/Gal4 transgenic D.melanogaster and its controls respectively in normal condition and under infection of different pathogen.The resuLts showed that the growth cycle,shape,weight,oviposition,pupation rate,hatching rate and life span of the UAS-wap/Gal4 transgenic flies did not obviously change under two kinds of cuLture conditions of normal and bacteria infection,but,control flies has extended their growth cycle by about four days,lost weight 23%,and reduced their oviposition by about 55%,pupation rate by about 60%,hatching rate by 20%,life span by more than 20 days post infection of pathogen.On the other hand,the immunity of UAS-wap/Gal4 transgenic flies was studied.The concrete contents also consisted of two parts.The first part was to detect the expression levels of wap and other antibacterial peptide genes in UAS-wap/Gal4 transgenic flies under the cu Lture conditions both normal and post-infection of pathogen by qRT-PCR.The second one was to do the statistics on the mortality and bacteria loads of UAS-wap/Gal4 transgenic flies and their controls post-challenged by bacteria.The resuLts of the first part showed that,under the condition of normal cu Lture,the expression level of wap gene in UAS-wap/Gal4 transgenic flies increased about 15 times more than that of their controls,and about 3 times than that of their own antibacterial peptide genes.But,the expression levels of their own antibacterial peptide genes had no significant difference between UAS-wap/Gal4 transgenic flies and the controls.Howere,post-infection by pathogen,the expression level of wap gene of UAS-wap/Gal4 transgenic flies rose precipitously,which was 80 times higher than that of the controls,and about 18 times compared to their inherent antibacterial peptide genes,while the expression levels of antibacterial peptide genes outside the wap in UAS-wap/Gal4 transgenic flies went up to 3 times compared to normal cuLture conditions,but no significant difference between UAS-wap/Gal4 transgenic flies and their controls.The resuLts of the second part showed that the mortality and bacteria loads were significantly lower in UAS-wap/Gal4 transgenic flies post-challenged in comparison to their controls.Two conclusions can be drawn from this study.First,the expression of wap gene has no effect on the physiological functions of UAS-wap/Gal4 transgenic flies,instead has enhanced effect.Second,the expression of wap gene has no effect on the expressions of their own antibacterial peptide genes,instead canimprove the immunity of D.Melanogaster.the first evidenceThis research has two significances.First,it is the first time to prove that the expression of wap gene is the main reason why UAS-wap/Gal4 transgenic flies have high antibacterial activities;Second,it is the first time to prove that the expression of wap gene is biosafety for genetically modified organisms because it did not change D.melanogaster's physiological functions and the expression levels of their own antibacterial peptide genes.Therefore,this research has provided a scientific basis for developing the Chinese shrimp WAP antibacterial peptide into drugs and for the application of genetically modified organisms.It has important economic and social significance to protect the security of human life and promote the development of animal breeding.