Expression Analysis Of Maize CMS-C Chimeric Genes Atp6-c、atp9-c And Cox2-c

Corn is the first crop to successfully utilize sterility seed production.Using sterile lines as female parents for sterility seed production can not only save the cost of artificial male removal,but also effectively improve the purity of hybrid seeds.Corn sterile cytoplasm can be divided into T（Texas）,S（USDA）and C（Charrua）.Corn CMS-T and CMS-S type sterile lines because there is disease,sterility relatively unstable defects and restricts their application in production,corn CMS-C because of the pollen completely,sterility performance is relatively stable,has important potential applications in hybrid seed production,but for the CMS-C type sterile lines abortive mechanism is still not clear.In this study,3 pairs of sterile lines and their maintainers and 3 groups were used as materials to analyze the spatial-temporal expression specificity of cms-c specific chimeric genes atp6-c,atp9-c and cox2-c,so as to systematically understand the intrinsic relationship between the chimeric genes atp6-c,atp9-c and cox2-c and the fertility performance of CMS-C.The main research results are summarized as follows:1.Anther expression analysis between sterile and maintainer lines at different developmental stages.Through fluorescence quantitative analysis,it was found that chimeric genes atp6-c,atp9-c and cox2-c were expressed in the anthers of maintainer lines,and normal atp6 genes were also expressed in sterile lines.When the anther length was 1.0～4.0 mm,the expression levels of atp6 in C Huangzao si and C48-2were significantly lower than those in Huangzao si and 48-2.However,the expression levels of atp6-c gene in C Huangzao si and C48-2 were significantly higher than those in Huangzao si and 48-2,with a maximum difference of 150,000 times.The expression levels of atp9-c gene in C Huangzao si and C48-2 were significantly higher than those in Huangzao si and 48-2.The expression level of cox2-c gene in anthers of C Huangzao si was much higher than that of Huangzao si.The anther length was 1.0～4.0 mm,and the expression level of cox2-c in C48-2 was higher than that of 48-2.Although it has been reported that the chimeric gene atp6-c does not exist in normal cytoplasm,it was found in this study that the chimeric genes atp6-c,atp9-c and cox2-c peculiar to CMS-C were expressed in microspores of maintainers at different development stages,and the expression level in sterile lines was higher than that in maintainers.2.Expression analysis of CMS-C sterile line and maintainer line.Through C Huangzao si and Huangzao si,C478 and 478 jointing stage and tasseling stage of root,stem,leaf and plant period of bract,the ear and filaments q RT-PCR analysis,found the atp6-c and atp9-c in C Huangzao si express the amount of the groups at different time points were higher than Huangzao si,including in jointing stage and tasseling stage expression quantity is higher than the root and stem leaf,the expression of the ear than bract and filaments.The results showed that chimeric genes atp6-c,atp9-c and cox2-c were expressed not only in the maintainers but also in other tissues.In different tissues at different periods,the expression level of sterile lines was higher than that of maintainers,and the expression pattern of atp6-c and gatp9-c enes was almost the same.3.Expression analysis of F₁in anther at different developmental stages of fertility restoration and fertility non-restoration.Fluorescence quantitative analysis showed that atp6-c gene was more expressed in F₁ with fertility restoration（C Huangzao si×Zi 330）than in F₁ without fertility restoration（C Huangzao si×18-599）during anther 1.0～2.5 mm.In the anther period of 1.0～3.0 mm,the expression levels of atp9-c and cox2-c genes in F₁ with fertility restoration（C Huangzao si×Zi 330）were higher than in F₁ without fertility restoration（C Huangzao si×18-599）.atp6-c,atp9-c and cox2-c genes had higher expression levels in F₁ without fertility restoration（C48-2×Zi 330）than in F₁ with fertility restoration（C48-2×18-599）in anther1.5～4.0 mm.In conclusion,the results showed that the chimeric genes atp6-c,atp9-c and cox2-c were expressed in different fertility and different periods,and the expression level of F₁ in which the father was from Zi 330 was higher than that in which the father was from 18-599 in the early anther development.4.Anther expression analysis of CMS-C restoration gene importing lines at different developmental stages.To avoid inconsistent genetic background affect on gene expression,a near-isogenic line introduced by the major restorer gene in the background of C Huangzao si was selected as the material,through a fluorescence quantitative analysis found that import after restoring genes,the fertility show the fertile anther BC₄F₁-F（2.0～2.5 mm and 3.0～5.0 mm）in atp6-c,atp9-c and the expression of cox2-c higher than that of sterility,show the sterility BC₄F₁-S However,the expression levels of atp6-c,atp9-c and cox2-c in BC₄F₁-F anther 2.5～3.0 mm were lower than those in BC₄F₁-S.The above results indicated that,in the case of relatively consistent genetic background,the chimeric genes atp6-c,atp9-c and cox2-c showed the same expression trend in different fertility and different periods,and there was no difference with the changes of F₁fertility performance.5.Prokaryotic expression analysis of atp6-c and atp6.In this study,p GEX-6p-1was used as a vector to construct the prokaryotic expression vector ATP6-N-6p of the normal gene atp6 and the prokaryotic expression vector ATP6-C-6p of the chimeric gene atp6-c,and the recombinant plasmid and the empty p GEX-6p-1 was transformed into E.coli BL21（DE3）,and the effect of the expression product induced by IPTG on the growth of the cells was observed.The results showed that the growth of ATP6-N-6p and ATP6-C-6p strains was inhibited by the strains containing recombinant plasmids ATP6-N-6p and ATP6-C-6p under the induction of 0.1 mmol/L IPTG,The inhibition of ATP6-C-6p was more obvious,and the growth of unloaded p GEX-6p-1 strain was basically consistent with the uninduced condition.These results suggest that the protein secreted by atp6-c may be toxic to cells.