Differential Proteomic Analysis Of Cytoplasmic-Nuclear Male Sterility Line And Its Maintainer In Wheat (Triticum Aestivum L.)

Wheat (Triticum aestivum L.) is the most important grain crop in the world.Heterosisutilization of wheat are important approaches to increase wheat yielding and quality.To betterutilize the wheat heterosis to serving for agriculture development,the mechanism of malesterility are the field of research focus for many years.Proteomics is one of the importantresearch of the functional genomiese.To better understand the molecular mechanism onprotein level and find the crucial proteins which related to fertillity,we use thecytoplasmic-nuclear male sterility line (S)-1376A and its maintainer line (A)-1376B as theplant materials,the differential proteomic analyses of anther and the mitochondrial proteomeof spike in wheat are systematically studied,the important results are as follows:1.We firstly optimized the two-dimensional electrophoresis conditions on the uninucleateanther stage of wheat,the results showed that,the proteins extracted by the TCA-acetonemethod,dissolved protein with the lysis bufferⅡ,with pH 4～7 srips,800μg loading quantityand 13% concentrations of the SDS-PAGE gel,the proteins were well separated,total601-631 protein spots were detected on the 2-DE gels map.The optimized condition of 2-DEwas suitable for proteomic analysis of wheat anthers.2.Total anthers proteins were extracted from uninucleate and binucleate stage of(S)-1376A and (A)-1376B for IEF/SDS-PAGE.An higher repartability and better separatingprotein method from wheat anther for 2-DE was established.The results showed that about610 protein spots could be visualized on the two dimensional electrophoresis map bycoomassie brilliant blue staining within Mr 9.0～100.0kD and pH 4～7,and about 94 spotswere differential expressed between male-sterile line and its maintainer line by PDQuestsoftware.Among those differential proteins,about 49 spots and 40 spots were differentialexpressed between uninucleate anther stage and binucleate stage respectively;Meanwhilecomparing with the 2-DE maps of the total proteins from the binucleate anther stage anduninucleate stage,15 differential spots were detectable at male-sterile line;and 16 differentialspots were detectable at its maintainer line.These diffence proteins which up or down-regula-ted protein and specific expressed or not expressed proteins between male-sterile line and itsmaintainer line maybe related with cytoplasmic male sterility of wheat. 3.A total of 28 differentially expressed proteins were identified by matrix-assisted laserdesorption ionization time of flight mass spectrometry(MALDI-TOF-MS),19 of them wereidentified following NCBInr database queries,which are ubiquitin-conjugating enzymeE2,putative glycine-rich protein,ascorbate peroxidase,putative cysteine proteinase inhibitor,ribulose bisphosphate carboxylase small chain clone 512,triosephosphate isomerase,two ofthem which were identified is 23KD subunit of oxygen evolving system of photosystemⅡ,4proteins were identified with a same protein-ribulose-l,5-bisphosphate carboxylase/oxygen-ase small subunit,putative fructose 1-,6-biphosphate aldolase,three hypothetical proteins andone unkonwn protein.These proteins were involved in the process of energy metabolism,protein degradation,cell defense,regulating flower development.These results provide a cluefor elucidating the mechanism of CMS.4.Plant mitochondria play a primary role of energy-conversion in a cell system,and alsoperform many other secondary functions including synthesis of nucleotides,metabolism ofamino acids and lipids,biosynthesizing vitamins and cofactors,meanwhile,mitochondria alsoplay a unique role in expression of plant CMS.Isalation of high pure mitochondria fromwheat is one of the key techniques in CMS mitochondria proteomic analyse.Mitochondriafrom wheat young spike were isolated by a differential centrifugation and Percoll density-gradient method.The three-step discontinuous Percoll density gradient centrifugation resultedin the formation of a mitochondrial ring at the interface of the 23% and 45% Percoll layers.Determined by marker enzyme assays and chlorophyll content,the mainly contaminants inthe spike mitochondrial fraction were caused by peroxisomes,plastids and chloroplasts whileetiolated seedling was free of chloroplasts contamination but with a slight content ofperoxisomes,plastids.Improvement in purity was obtained by a 28 %(v/v) Percoll self-forming density gradient centrifugation on the mitochondrial fraction from spike,these cont-aminants were decreased to negligible amount and the integrity of mitochondria (88%) wasimproved to 90%,and the mitochondria still kept activity well after gradient centrifugation.5.The large sets of soluble protein of the spike mitochondria was extracted for IEF/SDS-PAGE,the results showed that about 326 protein spots could be visualized on the twodimensional electrophoresis map by silver staining,It indicated that the protocol of puremitochondria isolation was found to be reliable and suitable for proteomic analyses for thespecific materials.Compared the 2-DE maps of the male sterile line and the maintainer line,11 spots were differentially expressed,5 of them were up-regulated and 2 of them were down-regulated in the male sterility line of (S)-1376A,3 were specific expressed in the maintainerline of (A)-1376B,1 was specific expressed in male sterility line of (S)-1376A.The diffenceproteins which up or down-regulated protein and specific expressed between male-sterile line and its maintainer line maybe related with cytoplasmic-nuclear male sterility of wheat.A totalof 5 differentially expressed proteins were identified by matrix-assisted laser desorption ioni-zation time of flight mass spectrometry(MALDI-TOF-MS),only spot1,4 of them wereidentified following NCBInr database queries,are manganese superoxide dismutase(Mn-SOD),MnSOD is a nuclear-encoded antioxidant enzyme that localizes to the mitochondriaand is the major antioxidant defense enzyme in mitochondria to protect mitochondrialcomponents from superoxide liberated,the absence of this protein in male sterile linesuggested that the constitutive level of antioxidant capacity was impaired,which might berelated to the cytoplasmic sterility.