Cloning And Functional Analysis Of Recessive Genic Male Sterile Gene In Cabbage Line 83121A

Cabbage(Brassica oleracea L.var.capitata L.)is an important biennial vegetable,which belongs to the genus Brassica and the mustard family,Brassicaceae(Cruciferae).Strong heterosis on yield,quality,disease resistance and stress tolerance is displayed in cabbage.Heterosis breeding is the main way to improve cabbage varieties.Male sterile mutants play an important role in the utilization of heterosis in cabbage,and are also important materials to study the development and regulation of plant reproduction.83121 A is a spontaneous male sterile mutant identifed from cabbage.Genetic analysis showed that the male sterility was controlled by a recessive gene.In this study,the molecular mechanism of the male sterile mutant 83121 A in cabbage was studied by cytological and molecular biological techniques.The male sterility gene was cloned and the molecular markers closely linked to the male sterility were developed,which laid the foundation for its application in the breeding of cabbage.The main findings are as follows.1.The 83121 A line exhibited normal vegetative development and had fully opened flowers,well developed nectar glands,and normal stigmas.However,the anthers of 83121 A were smaller and had no pollen grains.The results of paraffin sections showed that the tetrad in 83121 A could be formed normally.The tapetum of 83121 A was strikingly degraded at the uninucleate stage.The development of microspores in 83121 A stopped at the uninucleate stage and was followed by breakdown.Scanning electron microscopy and transmission electron microscopy showed that the tapetum vacuolation was serious and the tapetum corpuscles could hardly be detected in 83121 A.Moreover,microspores of 83121 A could not form pollen exine after being released from tetrad.We hypothesized that the defect in 83121 A pollen development is primarily due to the lack of exine formation.2.The gene expression of 83121 A and its near-isogenic fertile line 83121 B buds before binuclei microspore stage was analyzed by RNA-seq.A total of 2881 differentially expressed genes were obtained,of which 1310 were up-regulated and 1571 were down-regulated in 83121 A.By analyzing the differential expression of genes related to pollen exine synthesis,Bol023932,a homologous gene to CYP704B1 in Arabidopsis,was identified as an important candidate gene,which was named BoCYP704B1.The expression of this gene in 83121 A buds was severely inhibited.3.BoCYP704B1 was specifically expressed in the anther and tapetum of cabbage.The expression reached the maximum at tetrad and early monocyte stage,and then weakened to termination.Subcellular localization indicated that BoCYP704B1 was localized in endoplasmic reticulum.BoCYP704B1 shares high identity with CYP704B1 in Arabidopsis and CYP704B2 in rice.It is known that both CYP704B1 and CYP704B2 catalyze the hydroxylation of medium-long chain fatty acids.It is speculated that BoCYP704B1 may encode fatty acid hydroxylase,which is involved in the synthesis of pollen exine.4.Cloning and sequencing showed that a 5424-bp Ty3-gypsy type retrotransposon was inserted in the first exon of BoCYP704B1 in 83121 A.The retrotransposon insertion in BoCYP704B1 not only blocked the gene expression,but also changed the structure of the encoded protein.Unlike wild-type cabbage,the protein encoded by BoCYP704B1 in 83121 A could not perform the original biological function.5.Molecular markers which were completely linked to the male sterility in 83121 A were developed based on the mutation of BoCYP704B1.Linkage analysis showed that the homozygotic mutational BoCYP704B1 always cosegregated with male sterility,which means BoCYP704B1 corresponded to the male sterile gene and affected the male fertility in cabbage.These markers can be used for auxiliary selection of male sterile materials.Furthermore,it was proved that BoCYP704B1 from wild type could rescue the male sterility and was treated as the restore gene for the 83121 A mutant.We can use BoCYP704B1 to create maintainers for the male sterile line 83121 A by genetic engineering.6.Label-free quantitative proteomics was used to analyze the differential proteomics of 83121 AB buds before the microspore binuclei stage.A total of 1245 protein species were identified with significant differential abundance,of which 648 protein species were down-regulated in 83121 A.These differential proteins were mainly involved in pollen wall synthesis,fatty acid metabolism,amino acid synthesis and protein processing modification.These results suggest that these metabolic pathways play an important role in the reproductive development of cabbage.