The Expression Analysis Of The Genes Associated With The CMS-C In Maize

Cytoplasmic male sterility (CMS) plays an important role not only in the maize commercial F1 hybrid breeding programs but also in the research of interaction between cytoplasm and nuclear. CMS-C is one of the most attractive sources of male sterility for hybrid maize production for its advantage of disease resistance and stable fertility. However, the abortion and fertility restoration mechanism of CMS-C is not clear.The three special chimeric genes (atp9-c、coxⅡ-c and atp6-c) have been found in CMS-C maize. Unfortunately, it seems difficult to prove that they are responsible for the male-sterility phenotype. In this study, three genes atp9-c、cox2-c and atp6-c were considered as probably candidate genes for CMS-C. The materials include the C-type male sterile lines and maintainer lines, fertility restored F1. Identification of these genes was conducted from DNA or RNA level by PCR or RT-PCR. As for the key candidate genes, the processing for the transcripts such as RNA editing, RNA splicing were investigated. The main results are follows:1. The expression level of CMS-C associated genes was analyzed by real-time quantitative PCR. The genes atp9 and cox2 were almost all high expression at the tetrad stage, but low expression at the uninucleate stage. It may be related with the character of the abortion of CMS-C. The expression of atp6-c in the fertility F1 hybrids was lower than the CMS lines, and the difference were more than two folds. It indicated that its expression is reduced by the nuclear background of F1 hybrid which may be the one of the most mechanism of the fertility restoration of CMS-C.2. According to the sequence of mitochondrial genomes in maize of CMS-C and normal cytoplasm, the mitochondrial genes of atp6-c and atp6-n were cloned in CHuangzaosi, C48-2 and Huangzaosi,48-2. We found that the atp6-c existed in the DNA and cDNA of CHuangzaosi and C48-2. It was not found in Huangzaosi and 48-2. The atp6-n was detected in Huangzaosi and 48-2 at the DNA and cDNA level. It was interesting that the atp6-n was not detected in CHuangzaosi and C48-2 at the DNA level, but it can be found in CHuangzaosi and C48-2 at the cDNA level. The sequence analysis indicated that the sequence of amplified fragment in the mRNA of CHuangzaosi and C48-2 was highly homology with atp6-n. So it was named as atp6-n’. It was found in the mRNA of fertility restored F1. The structure characteristics of atp6-n’was analyzed and the homologous sequences were found in the mitochondrial genome in C-type cytoplasm. The 56Obp in front of atp6-n’ was same with the cox2 5’UTR. The 800bp at the back of the atp6-n’was same with the back of atp6-c. These fragments are scattered and interspersed with other genes such as cox2 in the mitochondrial genome in C-type cytoplasm. Further more, the transcript of atp6-n’may be yield by undergo trans-splicing. The cox2 gene-135 to-8 may be as the donor and the atp6-c+480 to+607 as the acceptor of atp6-n’.3. In this study, the RNA editing was found in atp6-n. Therefore, RNA editing of atp6 gene conservative area, the area of the+445 to+1280 of atp6-c and the area of the+385 to+1224 of atp6-n and the area of-135 to-8 of cox2 were analyzed by direct sequencing.18 editing sites were detected out in front of the there region.12 occurred at the second position of codon,4 occurred at the first position of codon,2 at the third position of codon. The new stop codon was created at the 18th site. All of the sites are conversion of C-U. Moreover, the new editing sites a, b were only found in the area of the 850bp of atp6-n. The editing sites a, b were only existed in CMS-C line and fertility restored F1. The editing sites a, b are conversion of T-C and G-A. The amino acid was not changed by the editing site a. But the editing site b caused positively charged lysine into a negatively charged glutamic acid.The editing frequency of a, b in fertility restored F1 was higher than in CMS-C lines at the tetrad and uninucleate stage. In the fertility restored F1 (C48-2×18-599white), the editing frequency of a, b was higher 31% and 27% respectively than in C48-2 at uninucleate stage. The editing frequency of a, b in fertility restored Fl (CHuangzaosi×Mol7) was higher 25% than in CHuagnzaosi. It may be associated with the fertility restoration.The editing sites of a, b in cox2 5’UTR were not found. The sites of a, b in cox2 5’UTR were C, A which were same as the atp6-n. Furthermore, one editing site in this region is same with the first editing site in the other three regions. However, the editing frequency of this site is lower than in the other three regions. It may be associated with the process of trans-splicing.4. The functional characterization of atp6-c was discussed through prokaryotic expression. The atp6-c was cloned into pET-28a, pGEX-6p-1. The vectors were transformed into E.coli Resseta (DE3) and induced to expression protein and make bacterial growth curve until E.coli grown to mid-log (OD600=0.5). We observed that E.coli contained pET-28a-atp6-c had a decrease in cell density while the target protein was too less to detect it by SDS-PAGE. E.coli contained pGEX-6p-atp6-c have not impacted the growth of cells. The possible reason is that the ATP6-C protein was expressed with GST through the pGEX-6p-atp6-c vector.According to the results, one hypothesis would be made about the mechanism of CMS-C in maize. In the one hand, the complete transcript of atp6-n was found in CMS-C lines. Unfortunately, the transmembrane domain of ATP6-C protein is similar with ATP6-N. It may interfere with the normal function of ATP6-N. ATP6-C may be a toxic protein. It was verified that it inhibited the growth of E.coli contained pET-28a-atp6-c. It may cause the anther abortion by affecting the permeability of the mitochondrial membrane. Correspondingly, the nuclear back ground of fertility restored F1 improved the expression of the atp6-n which would provide more energy for the development of microspore. In order to increase the expression of the atp6-n, the atp6-c was consumed greatly as the receptor of atp6-n’. The interference with the atp6-c and atp6-n maybe reduced and the toxic will be mitigated. All of these restored the fertility of C-type cytoplasm.