Separation, Identification And Analysis Of Differential Expressed Proteins Of Wheat BNS Male Sterility Line Based On ITRAQ Method

BNS (Bai-Nong Sterility) male sterile line, a new type of male sterile lines, possessing goodsterility and transformation, that was found and cultivated in2003by professor Ru Zhengang, a wheatbreeding expert, is of important value for the production of hybrid wheat. In order to study the sterilemechanism of this sterility line, three parts of researches were done in the paper:(1) The stability andvariability of BNS sterility was observed in2012;(2) All differentially expressed proteins was separatedand identified by means of iTRAQ combined with two-dimensional liquid chromatography tandem massspectrometry, taking anther as materials in which microspores development were at the stage of tetradphase, uninucleated period, and binucleated period．(3) The differentially expressed proteins function andmetabolic pathway was analyzed and annotated by using of bioinformation method. The main results are asfollows:1. When compared fertile pollen rate and self-seeds rate each period in2012with that of2008and2009, it were showed that the fertile pollen rate in2012rose by0.46%~19.94%, part of which reachedto significant level; and self-seeds rate in2012increased by2.74%~34.59%, and also reached tosignificant level. It was also found that, from correlation analysis between the sliding accumulatedtemperatures of per four days of32days before anthesis and fertile-pollen rate and self-seeds rate, the BNSfertility conversion on the temperature could be affected significant in most days from antherdifferentiation to pollen development, until four days before anthesis. The meteorological data in the past5years showed that the year of2012was a relative higher temperature one in this area. The lowesttemperature in April of2012there was higher1.13-3.88°C than that of in the past5years. It was the highertemperature that resulted in BNS’ infertility reduced and self-seeds rate increase in2012.2. The results of proteomics by iTRAQ showed that2305peptide fragments that matched wheatprotein library were separated, and283anther proteins were identificated. When the value of differentialexpression multiplied log2was above1.0and the value of P was below0.05,83kinds of differentiallyexpressed proteins were identified from tetrad and mononuclear period, and82from binucleated periodbetween sterile line and conversion line,83and81kinds of differentially expression proteins from sterileline and conversion. This differentially expressed proteins ratio in total protein29.33%、28.97%、28.62%,respectively. This is the first total proteins table and differentially expressed proteins table of pollen development from tetrad period to binucleated period in wheat BNS.3. Hierarchical clustering, GO analysis, metabolic pathways and interaction network analysis forthe differentially expressed proteins were carried out by use of the resources of network database. Theresult showed that the protein expression patterns in uninucleated period and binucleated period underfertile conditions, and uninucleated period under sterile conditions were similar. A greatest distance andlowest similarity were found between tetrad phase S1and binucleated period S3. GO and KO analysisshowed that differentially expressed proteins were mainly distributed in the energy metabolism. Interactionnetwork analysis of differentially expressed proteins showed that56of83had GO annotation in biologicalpathways. According to the Cytoscape’s results, differentially expressed proteins were also mainly relatedin biosynthesis of energy metabolism and regulation metabolism.Based on the results and analysis above, it is concluded that the variability of BNS sterilitybetween different years was the result of temperature effects, and the thermo-sensitive characters of BNSsterility is stable. The BNS sterile metabolic pathway was conjectured as follows: lower temperature couldaffect on the BNS sterile gene(s), which could send out apoptotic signals, leading to the cell transmissionpath blocked. And the sterile factor(s) may regulate the energy metabolism proteins which made energysupply shortage, followings related metabolic disorders, such as protein biosynthesis, carbohydratemetabolism, lipid metabolism, starch synthesis and so on. Finally, those metabolic abnormalities wouldresult in cellular component decomposition and pollen sterility.