Molecular Mechanism Analysis Of Maize CMS-C Mitochondrial Sterility Gene Atp6c

Male sterility seed production is an effective means to promote the utilization of heterosis in maize,and cytoplasmic male sterility(CMS)line is a powerful tool to utilize heterosis and study nucleocytoplasmic signal transduction and interaction.Maize type C CMS(CMS-C)has been widely used in seed production,but its sterility gene and its mechanism of action are still unclear.In this study,atp6 c was identified as the sterile gene of maize CMS-C by genetic transformation method,and the molecular mechanism of pollen abortion caused by sterile gene atp6 c was analyzed by cytology,molecular biology and other techniques.The main conclusions of this study are as follows:1.Maize C-type CMS line Yu87-1A and maintainer line Yu87-1B had no significant difference in the phenotype of each tissue before flowering;the maintainer line anthers gradually enriched from the S10 period,and the volume continued to expand,and the mature anthers were velvety.The felt layer degenerates normally,the pollen is scattered in the anther chamber,and the starch is fully dyed;the anthers of the sterile line gradually shrink from S11 until they become shriveled.From S10,the tapetum swells and becomes vacuolated,and the disintegrated microspore residues are close to the velvet.On the felt layer,the anthers were not exposed at the pollenscattering stage,the anthers were relatively small and shriveled,and the tassel had no pollen scattered and no starchy mature pollen grains.2.Physiological index determination showed that ATPase activity,ATP content,mitochondrial membrane potential and POD enzyme activity in Yu87-1A anther were relatively low,superoxide anion(O2-.)content,hydrogen peroxide(H2O2)content,SOD enzyme activity and MDA contents were relatively high;the abnormal physiological state led to the premature occurrence of PCD in sterile line anthers,and the duration was longer.3.Part of the atp6 sequence in the front part of cox2-2 in the sterile line is transcribed but does not affect protein translation and accumulation;genetic transformation of mts-cox2-2 vector does not produce pollen abortion phenotype;transgenic offspring of mts-atp6 c vector homozygous plant ATP6 C More protein accumulation can produce pollen abortion phenotype,and the plant phenotype is similar to Yu87-1A,and the trend of physiological indicators is also the same.4.Gene structure analysis showed that the 809 bp sequence between atp6 c and atp6 was only 2 bases apart.The first 39 bp of the coding region of atp6 c was derived from the first part of atp9 gene,and there was a single base variation;there were 18 c DNAs in both genes.RNA editing site,the last editing site produces a stop codon;protein high-level structure analysis shows that ATP6 and ATP6 C only have a transmembrane structure in the latter common amino acid sequence,and the N-terminal specific amino acid changes the spatial structure of the protein.A very low amount of atp6 gene exists in the Yu87-1A mitochondrial population,and its transcripts are free of RNA editing.5.atp6 c and atp6 were constitutively expressed in their respective materials and reached the highest level at S8b;the specific accumulation of ATP6 C protein in anthers was associated with the formation of sterility.6.The accumulation and enzyme activity of complex V in Yu87-1A were decreased,while the accumulation of F1′ was significantly increased.The enzyme activity of complex V was correlated with the accumulation amount.The results of western blot on collagen sites were consistent with the detection results of complex V activity in the BN-PAGE gel.The second-demention protein hybridization showed that the accumulation of ATP6 C was significantly increased in F1’ of Yu87-1A.The protein interaction study showed that there was no obvious interaction between ATP6 and ATP9/ATP8,and ATP6 C showed strong interaction with ATP9 and ATP8,and the interaction region was the specific region of ATP6 C.7.Gene detection of different subclasses of C cytoplasm shows that different subclasses have the same infertility genes,and the classification of cytoplasmic subclasses can be cancelled accordingly.Traceability analysis showed that the sterility gene only existed in the mitochondria of modern cultivated maize,and the corresponding c DNA was not edited.