Cloning, Prokaryotic Expression And Purification Of TaPDK Gene In Wheat (triticumaestivuml.)

Many researches indicate direct correlation between energy metabolism and plant malesterility. Pyruvate is an extremely important fuel source in the energy metabolism pathway,whereas pyruvic dehydrogenase kinase (PDK) plays an important role in energy metabolicregulation of higher organisms. Firstly, we took wheat leaf and anther from both sterile andfertile wheat varieties as experimental material, and tested their pyruvate content. Furthermore,a full length cDNA of TaPDK (Triticumaestivm pyruvate dehydrogenase kinase) gene wascloned to analyze protein-encoding features, function and protein-protein interactions. Aminoacid composition, conservational regions, phosphorylation sites and homology with othergenes were also analyzed. At last, a prokaryotic expression vector pET23d-PDK wasconstructed and the expression product was purified by nickel affinity column. The mainresults were as follows:1The determined results showed that pyruvate content reached to the minimum duringthe late mononuclear period, which probably resulting from pyruvate participation TCA cycle.This period is most likely to cause anther abortion.Befor the binucleate stage, pyruvate content was no significant difference in the anthersof three types of experimental materials. But at the trinucleate stage, pyruvate content inphysiologically male sterility declined sharply, It shows that energy metabolism ofphysiologically male sterility is much more susceptible. Low levels of pyruvate content mayaffect the normal metabolic pathway, which lead to the TCA cycle is blocked, insufficientenergy supply and pollen abortion2TaPDK gene is acquired and sequence analysisTaPDK ORF with the length of1095bp encodes364amino acids were obtained from thebinucleate stage of anthers cDNA. The minor codons content of25%. TaPDK proteinstructure and function are analyzed by bioinformatics, the results Show that it was located inthe mitochondria by Subcellular Location prediction, With a highly conserveddomain(HATPase_c). The domain contains13α-helices and8β-strands, bind ATP,andserine/threonine kinase activity3TaPDK prokaryotic expressionThe purpose gene was subcloned into the prokaryotic expression vector pET-23d(+) and constructing restructuring plasmid. The expression product whose molecular mass about40kD was checked by SDS-PAGE and Western Bloting, which showed target protein wassuccessfully expressed in E. coli BL21(DE3).4Purification of target proteinAfter nickel affinity column, the obtained fusion-protein was found to be composed of threebinds, the TaPDK protein and two stable nonspecific proteins. High quality TaPDK proteinwas obtained finally by cutting gel.Above all, this research which conducted a preliminary exploration of the TaPDK protein,established a prokaryotic expression system, and purified protein TaPDK. It laid thefoundation for polyclonal antibodies and studying protein function, and it is the foundation forstuding TaPDK protein in the role of energy metabolic regulation mechanism duringphysiological-type male sterility in wheat induced by SQ-1.