Differential Anther Mitochondria Proteomic Analysis Of Cytoplasmic Male Sterility Line P3A And Its Maintainer P3B In Kenaf(Hibiscus Cannabinus L.)

Cytoplasmic Male Sterility (CMS) is a widely biological phenomenon in higher plants. The molecular mechanism of CMS has not been clear figured out yet. It is generally believed that the mitochondrial genome recombinant to form abnormal open reading frames (orfs) is related to CMS. Proteins are the executor of the biological function, therefore, to carry out the mitochondrial proteomics research is of great significance as to explore the mechanism of CMS. In the present study, we used kenaf cytoplasmic male-sterile line P3A and its maintainer line P3B as materials. Two-dimensional gel electrophoresis (2D-PAGE) was adapted to study the dikaryophase anther mitochondrial proteins, gained the differences points of protein to do mass spectrometry. The atpl conservative region was cloned based on results of the proteins. We obtained the following results:1. Use IEF/SDS-PAGE Bi-directional gel electrophoresis technology, we obtain a clear gel electrophoresis of anther mitochondria proteins of kenaf CMS line P3A and its maintainer line P3B. In17-130kDa in molecular weight, isoelectric point3-10range, we can identify about1000protein points, ImageMaster2D platinum5.0software analysis results show that there are50variations of the mitochondrial proteins in male-sterile line and its maintainer line. Only7specifically expressed proteins in the male-sterile line and4in its maintainer line and other39for differences in quantity, there are23protein pionts are up-expressed in the male-sterile line and16in its maintainer line. 2.50different protein points with matrix assisted laser parsing ionization time of flight mass spectrometer with peptide fingerprint analysis (MADIL-TOF-TOF-MS), we get the28fingerprint of protein peptides and blast them with data in the Mascot database, we successfully identified24proteins. They are ATPase subunit B, beta-tubulin18, alpha-tubulin4, ATP synthase subunit alpha,the mitochondrial outer membrane protein,3proteins were identified as the same protein of ATP synthase F1subunit1, ATP synthase beta subunit, mitochondrial chaperone CPN60, peroxiredoxin-B,luminal-binding protein2, phosphoglycerate mutase,succinate dehydrogenase, two mitochondrial-processing peptidase subunit beta, mitochondrial-processing peptidase subunit alpha, malate dehydrogenase,mature K, cysteine-rich repeat secretory protein38,3hypothetical protein and one predicted protein respectively. These proteins mainly involve the energy metabolism,probably relates to the occurrence of a kenaf cytoplasmic male sterility (CMS).3. By homology cloning method, we designed specific primers with conservative region of atpl gene of other species in the Genbank, and obtained atpl gene1045bp sequences from the CMS line P3A and its maintainer line P3B of kenaf using PCR amplification. Compared the sequence show that there are few differences between each other, just seven nucleotides different.Respectively,P3A in the third place changed from A to G, in the58th/616th/1002nd places changed from C to T, in the685th place from T to C, in the786th and971st places from G to A; while P3B hiatus a nucleotide C in the21st place and a G in the1010th place,The homology was99.14%.The results of semi-quantitative RT-PCR showed that the expression level of atpl gene was not obvious difference between the CMS line and its maintainer line.