Cloning And Transcript Expression Of Candidate Gene Of CMS-S Restorer-of-Fertility Mutants Rfl~*04-229 And Rfl~*04-230 And The Preliminary Identification For A GMS Mutant K305ms In Maize

Maizeis a major crop in the world.The utilization of heterosis is one of efficient approach for improving maize yield.The happening of male sterility(MS)adversely impacts plant offspring reproduction,however,MS phenotype provides a valuable material for more widespread utilizationof heterosis,exploration of the mechanisms of pollendevelopment and interaction between cytoplasm and nuclear.In this study,the new restorer-of-fertility(rf)mutantsrfl*04-229 and rfl*04-230isolated from the maize cytoplasmic male sterility-S(CMS-S)mutant lines by Mu transposon insertion were selected as the materials.The candidate rf genes were identified by the Mu-Illumina sequencing technology.The phenotype,genotype,genes transcription were investigated to hypothesis the mechanism of fertility restorationfor maize CMS-S.Meanwhile,a male sterile mutant K305msinuced by Co60-?irradiation was selected as the material.The phenotype,genetic characteristics and metabolism of K305msmutant were preliminary identified.The main results are as follows:1.Two new rf mutants,rfl*04-229and rfl*04-230,rescued CMS-S collapsed pollen but conditioned a homozygous-lethal seed phenotype.2.The Mu-Illumina sequencing and BLAST analyzing(conducted by collaborators Alice Barkan and Rosalind Williams-Carrier at the University of Oregon)indicated that the Muinserted intoa mitochondrial Ribosomal protein large subunit 6(Rpl6)gene5'UTR in chromosomal 4 in rfl*04-229mutant.In addition,in rfl*04-230 mutant,the Muinserted into both a mitochondrial Ribosomal protein large subunit 14(Rpl14)gene and a gene encoding a member of the Oxa1/Alb3/YidC membrane protein insertion chaperone family(Alb3)in chromosomal 3.Maize mutant alleles ofrpl6,rpl14 and alb3 were proposed as the newly discovered rf candidate genes for CMS-S system compared with the other reported rf genes.3.The gene segregation and recombination was studied by crossing the rf#l(?)04-230 mutant with Mo17-N.Four individual of single mutant rpl14and three individual of single mutat alb3 were isolated based on PCR identification.The restored pollenrate ofrpl6(rfl*04-229)and rpll4 mutants were found to be approximately 50%by carmine acetatestaining.Whereas,alb3 mutant showed lower efficiency for pollen restoration,only restoring 20%of pollen sterility.4.Gene transcription patterns of Rpl6 and Rpll4 in the restored pollen of rpl6 and rpl14 mutants were detected by RT-PCR.The decreased transcript accumulation and an increased length of the transcripts'UTR were found in rp16 mutant,whereas a nearly undetected transcription level of Rpl14 was detected in rpl14 mutant.These results implied that the Mu insertion might block the synthesis of mitochondrial ribosomal protein subunit,it further inhibits the synthesis of mitochondrial genes encoded proteins(including the sterility related CMS-S protein),thereby rescued the collapsed pollen at translational level.5.The lethal seed phenotype was found in both progenies of sibing of rpl6 and rpll4 mutants,and theratio of lethalseeds and viable seeds,were calculated as 1:1.The abnormal phenotype of embryo and endosperm in lethal seeds of rpl6 and rpl14 mutants at 16d after pollination were observed by microscopic observation.Additionally,the amounts of basal endosperm transfer layer(BETL)were largely decreased,and the plasmodesmata could not be observed(or only little could be observed)inBETL.These results indicated that Rpl6 and Rpl14 gene played important roles in the development of BETL.Maize rpl6 and rpl14 mutants afford important materials for the further exploring of pollen and seed development.6.No phenotype difference was observed between maize K305ms mutant and fertile plants K305F at vegetative stage.However,K305ms mutant showed unexposed anthers in tassels,withered anthers and aborted pollen without function at reproductive stage.K305ms mutation type was found to be the male complete sterility based on I2/IK staining.The sibling progeny of K305ms mutant and K305F showed a fertility separation ratio of 1:1,and the self progeny of K305F showed a sterile and fertile separation ratio of 1:3 regardless of locations,years and seasons.In addition,the crossing K305ms mutant with four inbred lines showed 100%fertile pollen in all hybrids,implying that K305ms mutant was "non-pollen type" of genic male sterility controlled by a recessive single gene.7.The soluble protein content and free proline content in anther and glume of K305ms mutant was lower than K305F during pollen development,inferring that the protein and proline metabolism are disordered in K305ms mutant during pollen development.The activities of SOD and CAT in anther of K305ms mutant were significant or extremely significant lower than K305F,whereas the POD activity was significant or extremely significant higher,indicating that abnormal ROS metabolism is occurred in K305ms mutant during pollen development.