Proteomic Analysis On Avian Eggshell Quality And Inhibitory Expression Trial On Ion Transporter Genes

Eggshell strength is a crucial economic trait for table egg production. The genetic improvement of eggshell quality is efficient approach to reduce the losses caused by cracked eggs. Avian eggshell is formed in uterus with saturated inorganic ions and organic matrix protein. The interaction the these two major components determine eggshell quality. The ion transpoters in uterine epithelial cells can supply ions such as Ca2+and CO32-for the eggshell calcification, and the organic molecules in uterine fluid can regulate the process of eggshell calcification influencing crystal size and eggshell strength. this thesis carried out two studies related to the process of eggshell calcification.1) the potential roles of uterine fluid and eggshell matrix on eggshell calcificaiotn and eggshell quality;2) the inhibitory expression analysis of ion transpoter genes in uterine epithelial cells.(Part I) The screening of candidate proteins in uterine fluid and matrix related to eggshell qualityAccording to the investigation of eggshell formation to find the crucial genes or proteins related to eggshell quality.this is meaningful for the research of genetic mechanism of eggshell quality.In this study, a label-free mass spectrometry-based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs(SM-Strong and SM-Weak) and between the corresponding uterine fluids bathing strong and weak eggs(UF-Strong and UF-Weak) in a chicken population. The special strong and weak eggs differ significantly in eggshell strength, but not in eggshell thickness and eggshell weight.Here, we reported the first global proteomic analysis of uterine fluid. A total of577and466 proteins were identified in uterine fluid and eggshell matrix respectively. Out of447identified proteins in UF-Strong, up to357(80%) proteins were in common with proteins in UF-Weak. Similarly, up to83%(328/396) of the proteins in SM-Strong were in common with the proteins in SM-Weak. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, fifteen proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.(Part II) The inhibitory expression analysis of ion transpoter genes in uterine epithelial cells.The current study selected8genes related to the transportation of Ca2+, HCO3-, Na+, Cl-for the expression analysis after the treatment of acetazolamide and calcium restriction. This supply novel insight for the research of the interactions or interregulations among ion transporter genes.Twenty-two43-wk ISA Brown hens were used in this study, eight hens were treated with oral acetazolamide, and8hens were treated with calcium restriction(reduce the calcium concentration in feed from3.5%to1%), the rest8hens were considered as control group. Tissue samples (magnum, uterus, duodenum) were harvested in24hens while the egg was in the uterus during the active phase of calcification (16-18hour post-ovulation). Then RT-PCR were used for the detection of expression of ion transporter genesThe results showed that the treatment of acetazolamide and calcium restriction can sigficantly reduce eggshell weight(P<0.05), indicating the disorder of ion transpoter system in uterus after the treatments. According to the results and literatures several conclusions were drawn.(1) there exist different regulation mechanism for the Plasma membrane calcium-transporting ATPase1and2(ATP2Bland B2). The expression of ATP2B2was Ca2+concentration-dependent, while ATP2B2was more like feedback regulation, which may be regulated by hormones;(2) the expression of CALB1in uterus is affect by Ca2+concentration, but not affect in duodenum;(3) acetazolamide can significantly enhance the expression of CA2;(4) the low concentration of HCO3-in uterine epithelial cells can promote the expression of SLC4A4gene. The current study is the preliminary investigation for the interactions or interregulations among ion transporter genes.