Dynamic Analyses Of Proteins In Humor Of Embryonic Chicken

Embryonic chicken is a good model for the study on the embryo development for vertebrates. Fertilized egg can develops into a lovely chick just in 3 weeks, during the development, all nutrients are derived from the yolk sac and egg white, only given with a proper temperature and humidity, which exhibits a highly skillful ability for survival gleaned from evolution. With the annotation of chick genome in 2004, it is possible to carry out the functional genome research. We are interested in the dynamic pattern of proteins in humor of embryonic chicken, and hope to find a good aspect to participate the chicken functional genomics research.SDS-PAGE was used to survey the protein contents in allantoic fluid, aminotic fluid, and fluid-in-eye for 9-17days embryonic chicken. And then fluid-in-eye was analyzed with 2-D electrophoresis for 9-21days embryonic chicken, some protein spots were identified by peptide mass fingerprint obtained with MALD-TOF Mass spectrum, and the expression patterns of these identified proteins were discussed in this paper.1. For allanotic fluid, 6~9 main protein bands were observed without considerable changes between 9-17 days embryonic chicken, and its protein concentration is in low level. For aminotic fluid, traces of protein were observed in 9 days embryonic chicken, but increases with development, and from 13 days, the protein contents jump to high level, especially to 15,17 days, Which is consistent with biological events occurred in the development of the embryo that high quantity of proteins enter into aminotic fluid, which symbolizes that the embryonic chicken step into the fast-growing stage.2. For fluid-in-eye of embryonic chicken, which is mainly composed of aqueous humor and soluble components inside the lens, proteins pattern vary obviously from 9 to 17 days with quite high concentration and abundance. For samples of 15,17 days, the quantity of protein increases acutely, which seems to prepare for the vision formation. Results for 2-D electrophoresis of fluid-in-eye: there are fairly a sum of proteins showing different expression patterns during embryo development, which are believed to participate in eye development and vision formation directly. Two clusters of proteins were observed, the first clusters (S3 ,S8,S9,S12)MW is around 20kDa., the second (S10,S11,S13,S16) is about 25kDa. With the development of embryo, these proteins spots begin to appear, and their accumulation level increases, and reach a high quantity till 17 days and sustains the high level later, which suggest that these two clusters proteins were key participants invision formation(these two clusters were later identified as crystallins by mass spectrum). It is nearly the same case for Band 1(B1), which was formed across pH 3-10 in the gel for samples of 11 days, originally as some distributed spots in samples of 9 days, light density of Band 1 increases with development of embryo, and reaches high value at 17 days and sustains later. SI, S4, S5 were only observed in fluid-in-eye for samples of 9 days, which may be temporal-specific proteins. We also found a group of basic proteins with MW less than 14kDa, clustered together near the negative terminal IPG strip, which was labeled as Group A proteins. It is also interesting to find another proteins Band2 (B2) in the 2-D gel for the samples of 15 days and 17 days, which is not existent for samples of 9 days, 11 days, 13 days. More interests are that light density for B2 drops rapidly for samples of 19 days, and nearly disappears in samples of 21 days.3. 29,468 protein items were downloaded from NCBI and a trpsin digested mass searching library was established. About 15 protein spots from fluid-in-eye were treated for peptide Mass fingerprint determination. After the searching and identification, 8 spots were identified as crystallins, which were classified into two protein family, alpha crystallin(including S3,S12,S8,S19) and beta/gama crystallin(including Sll,S13,S10,S16).4. So the expression pattern of these identified proteins was deduced: for the fluid-in-eye in 9 days embryonic chicken, alpha-crystallin chain A (S3) was firstly observed with low light density. For samples of lldays, in addition to alpha-crystallin chain A (S3 ), alpha-crystallin chain B (S8) is also present as well as Beta crystallin B2 (BP) (S10X beta A2 crystallin (S13> S16) . When it up to 13 days, the concentration for members of alpha-crystallin has reached a certain high level, but for members of beta/gama crystallin, the concentration is still in relatively low, which may be detennined by their two different expression regulation mechanisms and different biological function inside the lens. When embryonic chicken develop into 15,17 days, all crystallins identified nearly reach their highest level, and sustain later.In general, low level of proteins in allantoic fluid is coincidence with its function, containing final metabolites and providing water for later embryo development. For aminotic fluid, 13 days later, with the embryo enter into a fast-growing stage, the protein concentration in aminotic fluid increases acutely. For fluid-in-eye, with abundant proteins, and different proteins boast different expression pattern, and a cluster of crystallins identified by mass spectrum provide us a good foundation for further works in functional genome field.