Reserach On The Restoration Mechanism Of CMS-C Restoration Gene ZmRf5 In Maize

Maize is the most successful crop in the world in utilizing hybrid advantages,and seed production is an important factor restricting maize yield increase.Male sterility seed production is an effective means to ensure high and stable yields in maize.It can not only ensure the purity and quality of maize hybrid seeds,but also reduce the cost of manual dehazing in seed production.C-type cytoplasmic male sterility（CMS-C）in maize is currently the main type used in seed production,but its restoration mechanism is still unclear.Therefore,in depth exploration of fertility restoration genes and analyzing the recovery mechanism can provide a theoretical basis for the utilization of sterile seed production technology.The candidate gene was located in the 382 kb physical interval on chromosome 2 by map-based cloning technology and fine mapping.There are six candidate genes within the interval,through expression level and sequence analysis,it was preliminarily confirmed that Zm00001d007531 is the candidate gene for ZmRf5.On this basis,the function of Zm00001d007531 was identified through genetic transformation experiment of overexpression complementarity and genome editing,and allelic testing further confirmed that Zm00001d007531 is an allele of ZmRf5.Subsequently,the fertility recovery molecular mechanism of ZmRf5 was analyzed by cytology,molecular biology and other techniques.Using techniques such as cytology and molecular biology to analyze the mechanism of ZmRf5 restoring C-CMS.The main research conclusions of this study are as follows:1.Observation of anther phenotype and cytology of restorer line C6233R and sterile line CMo17A:The anther development of the restorer line C6233R gradually expands and becomes full and plump.The tapetum layer of the anther begins to degrade normally from the S12 stage,and the microspores gradually mature,starch accumulates and mature to form vibrant pollen grains;The anthers of the sterile line CMo17A start to become shorter and their morphology gradually shrinks from the S10 stage.The tapetal cells begin to swell and dilate from the S8a stage,and the microspores begin to degrade.The anthers are completely wrinkled,transparent,and dry,unable to form pollen grains.2.By analyzing the expression levels and sequences of six genes within the candidate interval,it was preliminarily confirmed that Zm00001d007531 is a candidate gene for the C-type cytoplasmic restoration gene ZmRf5.Zm00001d007531（ZmRf5）has a total length of 2445 bp and encodes a typical P subfamily PPR protein,containing 19 P motifs,targeting mitochondria.In the sterile line CMo17A,two large fragments,23 bp and 111 bp,are missing,causing premature termination codons and encoding only 37amino acids as a non functional peptide.Overexpression of Zm00001d007531^C6233R can restore the fertility of male sterile lines,while knocking out Zm00001d007531^C6233R can lead to loss of fertility in the restorer line.Transgenic functional complementary experiment and allele testing can confirm that Zm00001d007531^C6233R is the restoring gene ZmRf5 for maize C-type cytoplasm.3.The GUS staining experiment of Arabidopsis transformation showed that ZmRf5belongs to the constitutive expression gene;RT q PCR quantitative experiments showed that it is expressed in the roots,stems,leaves,and anthers of maize,and its expression level reaches its highest in the S6 stage of anthers.4.Zm RF5 promotes the cleavage of the 5’region of the full-length transcript of atp6c by transferring the cleavage site from 480th nt to 344th nt,ultimately resulting in a higher expression level of the total transcript in the restorer line C6233R compared to the sterile line CMo17A,while the expression level of the full-length transcript is significantly lower than that of the sterile line CMo17A.5.Western blot results showed that the accumulation of ATP6C in CMo17A was significantly lower than the accumulation of ATP6 in NMo17B and also lower than it in OE lines and hybrid CF₁ between restorer and sterile lines,was exhibiting a contrary trend in the Yu87-1 nuclear genome previously reported.The results showed that Zm RF5 restored the protein levels of ATP6/6C in CMS-C,and different nuclear genomes also had an impact on the expression of ATP6C protein.6.R-EMSA and Y1H assay confirmed that ZmRf5 does not directly bind to the transcript of atp6c.Two proteins,MORF8 and RS31A,that bind to atp6c were identified through RNA Pull Down.R-EMSA experiments confirmed that RS31A directly binds to atp6c,while MORF8 does not.Zm RF5 recruited RS31A,a splicing factor,through MORF8 to form a cleaving/restoring complex,which promoted the cleaving of the CMS-associated transcripts atp6c.7.The eighteen edited sites of atp6c RNA was detected,and it was found that 4editing sites were inhibited to varying degrees,but the 4 sites did not overlap with the cleavage sites,indicating that atp6c editing and cleavage are two independent processes.8.The accumulation and enzyme activity of complex V in restore line C6233R and CF₁ anther were restored compared to the sterile line CMo17A.The same results were observed in BN-PAGE gel western blot and the second demention protein hybridization test,as well as the accumulation of ATP6/6C and ATPβare much higher than that of sterile lines.The presence of Zm RF5 replenishes the amount and activity of complex V,and restores fertility.9.Physiological index determination showed that strain with ZmRf5 showed an increase in ATP content,H₂O₂ content,and MDA content.Although ATPase and POD activities were slightly lower,high activity CAT relieved stress and ensured the normal function of the tapetal cells.At the same time,the expression levels of family genes related to reactive oxygen species（ROS）clearance were higher than those of sterile lines and Crispr materials,which also alleviated pressure by clearing excessive H₂O₂,restored normal function of the tapetal cells,and restored fertility.