Exploration And Preliminary Identification Of Genes Related To Rn-type Cytoplasmic Male Sterility In Soybean

Plant cytoplasmic male sterility（CMS）is caused by sterile genes,which are produced by mitochondrial gene rearrangement,resulting in the plants incapable of producing or releasing pollen,but accept foreign pollen to complete the fertilization and produce offspring.With the discovery of CMS in many kinds of plants,sterility related genes have been identified.The cytotoxic mechanism,the energy deficiency mechanism,the abnormal programmed cell death mechanism and the retrograde regulation mechanism have been proposed to explain the molecular mechanism of sterility.In this paper,we observed the pollen fertility,plant phenotype and cell morphology of RN-type cytoplasmic male sterile lines and maintainer lines during their development.Mitochondrial genomes of male sterility lines and maintainer lines were sequenced,assembled and analyzed,and candidate genes of sterility were explored and preliminarily identified.The main results are as follows:（1）Comparing the male sterile lines with the maintainer lines,it is found that the sterile line RN-JLCMS9 A had highly sterile pollen,maintained vegetative growth,could not set pods,and did not shed leaves.Scanning electron microscope showed that the anthers could not crack and the pollen development was abnormal.Microscopic observation showed that the degradation of tapetum was delayed,and the sterile type was gametophyte sterility.（2）The mitochondrial assembly and annotation of male sterile line RN-JLCMS9 A and maintainer line JLCMS9 B were successfully completed.Obtained the complete mitochondrial genome sequence of 402 579 bp and 402 033 bp,respectively.Nine sterility candidate genes were screened,and the encoding products of five genes were predicted to contain transmembrane structures.（3）According to the mitochondrial differential sequence of male sterile lines and maintainer lines,molecular markers were designed to screen the RN-type male sterile cytoplasm of soybean.Some of the markers showed good specificity,which assisted in screening materials containing RN-type male sterile cytoplasm at the molecular level.（4）Quantitative Real-time PCR was used to analyze RN-CMS related mitochondrial functional genes.The results showed that there were significant differences in the expression levels of atp1 and atp8 between sterile lines and maintainer lines.It was speculated that the sterile genes may cause pollen abortion by affecting the expression levels of atp1 and atp8.（5）Prokaryotic expression vectors of five sterile candidate genes were constructed to study the effect of the expression of sterile candidate genes on Escherichia coli（E.coli）.The results showed that orf50 strongly inhibited the growth of E.coli,and it was speculated that the expression of sterile genes would produce toxic proteins to inhibit the growth of E.coli.