Differential Proteomics Analysis Of Flag Leaves Plasma Membrane In Physiological Male Sterile Wheat

Heterosis was the widespread natural phenomenon in plants. As one of the mostimportant cereal crop, wheat exist heterosis. It is difficult to using heterosis in wheat becauseof its complicated genetic background, but CHA-SQ-1provides a new method to solve thisproblem. The normal wheat can become that of male sterile when spray appropriate doses ofCHA-SQ-1at the right time, and proportion of male sterile wheat can reach95%~100%,outcrossing rate can exceed85%. Many years of manufacturing practice showed thatCHA-SQ-1of stability is high and super-high-yield wheat varieties have been obstainedthrough using CHA-SQ-1. Many significant achievements had been obtained to explainmechanism of male sterility induced by CHA-SQ-1. Differential proteomics of wheat flagleaves and differential expressed proteins were analysis in this study for providing moreevidence to explain mechanism of male sterility induced by CHA-SQ-1. Following wereachievements obtained through this study.1. Content of O2-was determined in wheat of different treatments (2hours,6hours,10hours12hours and24hours after spaying CHA-SQ-1). The results showed that content of O2-in wheat were increased significantly in ten hours after spaying CHA-SQ-1and began todecline at12hours after spaying CHA-SQ-1, eventually, returned to normal level after24hours. This result indicated that influence to wheat flag leaves induced by CHA-SQ-1is notcontinuous.2. A highly purified plasma membrane fraction was isolated by using differentialcentrifugation combined with aqueous two-phase partitioning [6.4%(W/W) PEG3350/Dextran T-500] from wheat flag leaves (Feeke，s8.5). The result of plasma membrane puritydetermination showed, the proportion of plasma membrane vesicle purified through aqueoustwo-phase partitioning reached87.9%, only50.1%compared to that of unpurified, andproportion of mitochondrial membrane and tonoplast contaminant were4.6%and1.3%, up to19.3%and7.53%compared to that of unpurified. The method of highly purified plasmamembrane isolation was established.3. PM proteins were deposited and washed by TCA/acetone and acetone. High-quality,good-repeatability2-DE maps were obtained through optimizing2-DE conditions, including choosing optimal PM protein loading quantities (900μg/gel) and gel concentration (12%). Thesystem of2-DE was suitable for plasma membrane proteomic analysis of wheat flag leaves.4. About200protein spots were distinguished on every2-DE map of wheat flag leaveswith spaying CHA-SQ-1(2,6hours after spaying) and no spaying CHA-SQ-1.20differentialexpressed proteins (total24proteins) were identified through MALDI-TOF/TOF MS analysiscombined with retrieving NCBI and SwissProt databases. These proteins were ATPasesubmits, FtsH, ASR protein, G protein,14-3-3protein，members of Hsp/Clp family andAGPPase and they participate in many metabolic process which might lead to male sterilityinduced by CHA-SQ-1, such as ion transport, coordinated regulation, signal transduction,starch synthesis and stress response.