Cloning And Structure Analysis Of Fertility-related Mitochondrial Genes Of Maize CMS-S

Cytoplasmic male sterility (CMS) is a kind of common phenomena in higher plants and is manifested as the inability of a plant to produce viable pollen; female fertility is unaffected. CMS is a: trait of economic importance in the production of hybrid seed as it eliminates the ineed for emasculation to pollination, thus reducing labor costs. A correlation between CMS and unique arrangements of the mitochondrial genome has been found. Nuclear festorer-of-fertility genes suppress CMS phenomenon to produce normal pollen.Eighteen lines of maize under Mo 17 and 77 nuclear background were used for mitochondrial RFLP, including N, T, C, S four types of cytoplasms. The plentiful polymorphism has been detected in most probe/enzyme combinations. Generally the RFLP pattern in one group is identical. But it is different from the others. The RFLP results can be used for classification of CMS lines quickly and also give us much more information about the structure of mitochondrial genes that may lead to CMS.In order to isolate mitochondrial genes easily, we set up a new method to construct CMS-5 group mitochondrial gene library by the means of embedding mitochondria and enzymatic digesting mitochondria in situ, the preparation of mtDNA by electrophresis, digesting LMP agarose by -agarase , using BAC vector and electroporation. It is also helpful to those who try to clone mitochondrial genes from other plants because it is more different to isolate mitochondria and mtDNA than from maize.A total of about 2,500 white clones of Mol7CMS-J mitochondrial gene library were obtained with the average size of ca 18.24kb, ranging from 5kb to 40kb, 63.6% inserts came from mitochondrial genome and represented 48X mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. Two contigs were revealed and showed the different structures of Mo17CMS-J mitochondrial genome from the CMS-SThrough PCR using artificial primes, 1kb DNA fragments were obtained from mitochondrial DNA of TangXu and WB cytoplasms. The DNA sequences are almost the same as the sequences in R region of CMS-S . Using the DNA fragment from TangXu cytoplasm as probe to scan the Mol7CMS-J gene library, several positive clones appeared. The clone B39 contains a 6.7kb BamHI fragment with R region. Wesubeloned this fragment and sequenced it Eive orfs including the chimeric orf77 were found in this area.Obviousdifferences of TangXu and shuartg cytoplasms in differentnuclear baekgrounds in Rregions were revealed, Homologous sequences of R werealso found in N and T groups, but not in C group. The R in N group was different from that in S group.T group had only part of R region. The heterogeneity of R regionsuggests that that the organization and rearrangement of mtDNA may be controlled by thenuclear background. Rregion seems to be omvolved in the mechanism of S group CMSand concerned with instability of S group.