Studies On Expression Stage, Position And Gene Functions Of The Selenoprotein G-rich Of Drosophila Melanogaster

Selenium is one of the essential trace elements for human body. Close relationships between selenium and many human diseases, such as cardiovascular, cancer and other diseases, are shown in many experiments. Selennoproteins are proteins in which the trace element Selenium inserted into proteins in the form of selenium homocysteine (Sec), by terminating codon UGA and special coding mechanism. Until now all of the selenoproteins whose molecullar functions are elucidated are important enzymes. So the studies on the functions of Drosophila selenoproteins have important significanceSelenoprotein G-rich is one of the three selenoproteins of Drosophila melanogaster. It is also the only selenoprotein whose function is still unknown in Drosophila. So the studies on the changes of the expression of G-rich at different development stages and different positions,and the variation of the expression of many other genes after the dsRNAi of selenoprotein G-rich are very useful for the elucidation of the molecullar functions of Drosophila selenoprotein G-rich.In this seperiment, the egg, first instar larvae, second instar larvae, third instar larvae, pupae and adult fly of Drosophila melanogester were used as samples for the studies of the mRNA expression of Drosophila selenoprotein at different developmental stages. The total RNA of the samples were were extracted and RT-PCR was carried out to study mRNA expression of Drosophila selenoprotein G-rich. And it was found that mRNA expression amount of Drosophila selenoprotein G-rich was highest in the stage of third instar larvae.In Situ hybridization was carried out to study the expression position of Drosophila selenoprotein G-rich in the embryo of Drosophila, using the whole embryo of Drosophila as samples and digoxin labeled antisense RNA of G-rich as probe. And it was showed that G-rich was mainly expressed in the Anterior midgut rudiment(amg),posterior midgut rudiment(pmg),mesoderm and midgut(mg) position of the embryo of Drosophila. And it was verified that the expression of G-rich was important for the development of the embryo of Drosophila.For the process of detecting the influence of G-rich on the expression of other genes the dsRNAi of G-rich and microarray detection were carried out. The RNA transcription template of G-rich with T7 promoter at both ends was first made by PCR, and double standerd RNA was transcripted in vitro by MEGAscript T7 Kit (Ambion) and anealed. The dsRNA of Drosophila was transfected into Drosophila S2 cell line by immersion method. After dsRNAi of G-rich, the totol RNA of dsRNAi treated and non dsRNAi treated Drosophila S2 cell line were extracted by Trizol reagent. The microarray was carried to study the changes of many G-rich related genes after the knowdown of Drosophila selenoprotein G-rich. And the gene functions of G-rich were analysed combined the microarray results and the results of the researches on the expression stage and expression position of G-rich. It was found that the expression amount of many genes related to anti-oxidization, protesis, anti-bacteria, programmed cell death, autophagy were elevated significantly. It was considered by most researchers that, after the knowdown of one gene, the expression of the gens processing the samilar functions normally will be elevated to make up its function lossing. So Drososophila selenoprotein G-rich was very possiblely having the anti-oxidization functions as the selenoprotein of BthD of Drosophila and the cellular glutathione peroxidase (cGPX), extracellular glutathine peroxidase (eGPX), phospholipid hydroperoxidase glutathione perxidase (PHGPX), gastrointestinal glutathione peroxidase (GPX-GI) and other selenoproteins in Homo.The microarray detection results of G-rich showed some degrees of similarity with the results of the expression of G-rich on the different positions of Drosophila embryo and on the different stage of Drosophila development. It was found that G-rich processing anti-oxidation effect by dsRNAi and microarray, and it was found that G-rich was mainly expressed in the positions of Anterior midgut rudiment(amg),posterior midgut rudiment(pmg),mesoderm and midgut(mg) of Drosophila embryo, which tissues are mainly developped into the alimentary system later. The anti-oxidation enzymes often expressed high in these positions found by other researcher. So the In Situ results could verify the anti-oxidation of G-rich. It was showed that G-rich mainly expressed in the third instar larvae stage in the studies on the expression of G-rich in the different development stages by RT-PCR. The volume of intestinal tissue was largest in the body in this stage pompared to almost all of the other fly development. This also verified the anti-oxidation function of G-rich indirectly. Above all it was concluded that Drosophila selenoprotein G-rich was a protein related to anti-oxidation in Drosophila melenogaster.