Molecular Biology Studies On C-Type Cytoplasmic Male Sterility Of Maize

Cytoplasmic male sterility (CMS) is the most important type of male sterility in maize (Zea mays L.), which occurs in many plant species. The investigation and utilization of CMS have been the mainly content in maize breeding. Cytoplasmic. male sterility in maize (Zea mays L.) is categorized into T-CMS, C-CMS, and S-CMS types based on the specific restoration of nuclear gene, in which C-CMS has a great application potential in maize hybrid breeding due to its stable sterility and resistance to Helminthosporium maydis Nisik. The sterile mechanism of C-CMS is not fully understood for its later discovery and utilization than these of T-CMS and S-CMS.Mitochondrial DNA extracted from two set of isonuclear alloplasm materials were analysed and compared each other with AFLP technique. One differential DNA segment of C48-2—named C48-223—was found when the mtDNA fragments were amplified with selective primers. The band was isolated , purified and sequenced. Based on the blasting result, the C48-223 is a part of atp6-C, which is a chimeric gene that uniquely associated with the C male-sterile cytoplasm of maize. The results of Southern blot indicate C48-223 is a real band. Via Northern blot, the signals can only be detected at the tetrad stage and uninucleate stage of anther RNA extracted from C48-2. Another differential DNA segment of C-huangzaosi—named C-huangzaosi1—was found when the mtDNA fragments were amplified with selective primers. Based on the blasting result, the C-huangzaosi1 is a part of NADH-C dehydrogenase subunit 1, which is uniquely associated with the C male-sterile cytoplasm of maize. All these indicate that the two fragments probably have strong relationship with the C male-sterile cytoplasm of maize.After optimized the 2-D system, we choosed the 2-D system for maize mitochondrial proteins: extract reagent contain 7.0 mol L^-1 Urea, 4%CHAPS, 2.0 mol L^-1 thiourea, 9.0mmolL^-1DTT, 0.8% ampholyte; 12% polyacrylamide gel; total 90000Vh isoelectric focusing; 500μg of protein extract per isoelectric focusing unit; 17cm immobilized non-linear pH gtadients(pH3-10). The mitochondrial proteins of maize yellow seedling, uninucleate stage anther , and binucleate phase anther of C48-2 and N48-2 (maintenance line) were separated by two-dimensional electrophoresis with immobilized pH (3-10, non-linear) gradients as the first dimension and SDS-PAGE as the second. The Coomassie brilliant blue-stained stained proteins spots were analyzed using PDQUEST software, there were about 280, 260, 220detectable spots on 2D-gel of respective phase. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 7 protein spots out of 23 were identified with significative data. They were F1-ATPsynthase, ATP synthase , Phosphatidylinositol 3-kinase , Glutamate dehydrogenase , voltage-dependent anion channel protein(two spots) and protein disulfide isomerase. F1-ATPsynthase and ATP synthase are relate with provide energy to cell. Glutamate dehydrogenase is present mainly in mitochondria in hight plants and play a important role in the course of nitrogen metabolism. VDAC plays an essential role not only in the permeability of the mitochondrial membrane, but also in apoptosis mediated by the mitochondrial pathway . Phosphatidylinositol-3 kinase (PI3K) is one of the most important regulatory proteins that is involved in different signaling pathways and controlling of key functions of the cell. The major role for protein disulfide isomerase is thought to be the formation and rearrangement of disulfide bonds.In summary, AFLP and 2-D dimention were employed to compare the mtDNA and mitochondrial proteins between the C type cytoplasmic male sterility and Normal cytoplasm of maize. This study will facilitate the understanding of the molecular aspects of the cytoplasmic male sterility of maize.