| Cytoplasmic male sterility (CMS) plays an important role in the heterosis utilization. In order to make better use of resources, select more stable CMS materials, the researchers expect to explore the mechanism of CMS abortion and fertility restoration in maize at the molecular level. People have engaged in studying the mechanism of CMS Abortion for a long time, it is widely considered that abnormal chimeric gene in mitochondrial genome formed by intramolecular or intermolecular frequent recombination is the molecular basis and primary factor of CMS. CMS fertility restoration is thought to rely on nuclear restorer gene, which can interfere with the activities of CMS-related chimeric gene and impact on transcript pattern, transcript abundance and transcript process of editing and translation of chimeric gene. However, so far, there are considerable limitations on understanding and utilizing of maize CMS-C, In order to provide the theoretical basis and application foundation for extensively utilization of C-cytoplasm in maize, this study focuses on identifying the mechanism of sterile and fertility restoration of CMS-C isoplasm allonuclear male sterile lines, The results are as follows:1. The experimental materials conclude isoplasm allonuclear male sterile lines:C HuangZaoSi, C48-2, C478; the testers:18-599White, Zi330 and the testcross progeny F1:(CHuangZaoSi×Zi330),(C48-2×18-599White),(CHuangZaoSi×18-599 White),(C48-2×Zi330), (C478×18-599White) and (C478×Zi330). Randomly selecting 200 pairs of SSR primers distributed in 10 chromosomes of maize, the differences in restoring and maintaining relationship between C HuangZaoSi, C478 and C48-2 were detected and analyzed at DNA level. It is shown hat primer AC204515.3-10 at the third chromosome amplified a single band with the same size 250bp in CHuangZaoSi, C478, C48-2, 18-599White and Zi330, male sterility F1 (CHuangZaoSi×18-599White), (C478×18-599White) and (C48-2×Zi330). While amplified a specific band slightly larger than 250bp in restored Fl generation (CHuangZaoSi×Zi330), (C478×Zi330) and (C48-2×18-599White). Therefore, it is suggested that the specific band produced by nucleus-nucleus and nucleoplasm interaction in fertility restoration F1 (C HuangZaoSi×Zi330) and (C48-2* 18-599White) may be associated with C-type cytoplasm fertility restoration.2. The experimental materials conclude two groups of isoplasm allonuclear male sterile lines, their corresponding maintainer lines and testcross progeny F1 of these two sterile lines, the transcripts of three chimeric genes (atp6, atp9, cox2) were analyzed by RT-PCR and sequenced during the tetrad stage and uninucleate stage. The results suggested that:the expression level of atp6 in the restored F1 was higher than in sterile and sterile F1. It is indicated that the atp6 is associated with fertility restoration. The expression level of atp9 in the two groups of isomer have their own characteristic expression, which seems negatively correlated with fertile. During the tetrad and uninucleate stages, the expression pattern of cox2 chimeric gene between two groups of materials was as follows:in the tetrad period, the expression level of sterile lines C HuangZaoSi, sterile Fl hybrids (CHuangZaoSi×18-599White), fertile hybrids (C HuangZaoSi×Zi330) was no significant difference. However, in the uninucleate stages, the expression level of fertile hybrids (CHuangZaoSi×Zi 330) was much lower than that in C HuangZaoSi and sterile hybrids crosses(CHuangZaoSi×18-599White). Meanwhile, in the tetrad and monocyte periods, the expression level of fertile F1hybrids (C48-2×18-599White) was positively lower than that in C48-2 and sterile Fl hybrids (C48-2×Zi330). The results indicated that the cox2 may be intimately related to the abortion of CMS-C.3. The analysis of specific mRNA sequence of cox2 revealed that there were two significative base mutation in fertile F1 hybrids (C48-2×18-599White), (C HuangZaoSi×Zi 330) and sterile F1 hybrids (C HuangZaoSi×18-599White), (C48-2×Zi330). At 159 and 279 sites, in the two fertile F1 hybrids were both A and C, while G and T in the sterile F1 hybrids. According to amino acid sequences alignment, it is found that these different sites produced only one amino acid alteration which occurs at 53 sites and resulted from the alterative base at 159 sites. In sterile F1, the amino acid is methionine (M, hydrophilic index of 1.9), while in two fertile combinations is hydrophobic strongest isoleucine (I, hydrophilic index of 4.5). The isoleucine has a large R group that would hinder the formation of a-helix, which maybe affect the stability of protein encoded by cox2. The amino acid sequence encoded by cox2 is quite similar with the part of amino acid sequence encoded by atp6, which may probably interfere with the normal function of atp6. Summary, the expression level of cox2 in fertile Fl is further decreased, and the structure of protein encoded by cox2 which involved in interfering with the normal function of atp6. Both above-mentioned phenomenons reduced the poisoning effect on atp6 protein in C-type cytoplasm and fertility is restored finally. |
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