| Cytoplasmic male sterility (CMS) is widely existence in higher plants. Study andapplication of CMS and its restoration (Rf), not only excuse us from artificial emasculationand contain potential capacity in crop seed production, but also provide a valuable researchmode for revealing the interactions between nuclei and cytoplasm. The polima (pol) CMS wasfound in Brassica napus, and has been widely used in Cruciferae, but the mechanism of malesterility was still unknown. In our team, the pol-like CMS has been successfully transferredinto heading Chinese cabbage, the restorer gene of which was also found in Chinese cabbage.In this research, we separated and identified six specific expressed genes between male sterileand fertile plants; screened the molecular markers and located the restorer gene BcRfp. Thedetail results were as follows.First, screening and identification of the reference gene that expressed most stable duringflower bud development in heading Chinese cabbage. Three statistical softwares were used tocompare the expression stablity of eight candidate genes in different sizes of buds. The resultsby GeNom software showed the order of expression stability was: Tub/GAPDH> Cyp>EF1a> U34559> BcTip41> Apr>18S rRNA, Tub and GAPDH were the most stablereference gene with the M value0.614. As the results of NormFinder and BestKeepersoftware, Tub was also the most stable reference gene. Comprehense three softwarecalculation results, Tub was regarded as the best reference gene, and could be used for thegene expression analysis during flower bud development in heading Chinese cabbage.Second, screening and identification of CMS and restorer related gene. CDNA-AFLPanalysis was used to identify genes differentially expressed during flower bud developmentbetween cytoplasmic male sterile plant (Ms) and male fertile plant (Mf) derived from thehybrid of CMS7311and a restorer (Rf). Thirty-two transcripts of different fragments (TDFs)of over80bp in length were identified by cDNA-AFLP anslysis. Among them, ten TDFswere found expressing in Ms bud, and the others expressing in Mf bud. Gene ontologyanalysis revealed that these genes were involved in stress response, synthesis and metabolism of fatty acid, cell reconstruction etc.Third, six genes closely related to male sterile were selected and their expression duringthe flower bud development of Ms and Mf line were investigated by qRT-PCR. Three genes,BcGRP17, BcMS2, and BcPME31, which were necessary for normal male organ development,showed significantly lower expressions in Ms bud; two genes, BcROPGEF8and BcTNL3,were induced significantly higher expression in Ms bud by unknown stress and involved inthe formation of male sterility. Another function unknown gene BcUP was only expressed atthe early and middle stage during the flower bud development,and the expression in Ms budwas obviuously lower that that in Mf bud.Fourth, clone of unknown gene BcUP. Based on the results of cDNA-AFLP analysis, afull length cDNA BcUP with1407bp was cloned by RACE method, which containing acomplete development reading frame1212bp and encoding a protein with403amino acidresidues. Blast result showed that this gene was homology with an unknown function geneAT3G56750from Arabidopsis, with the similarity about90%. A glycosyltransferase domainwas predicted by bioinformatics suggesting that this gene may have a fucosyl transferasefunction.Fifth, screening of molecular marker linked to the restorer gene BcRfp of CMS andmapping. By molecular marker techniques such as AFLP, SRAP, SSR etc, a series of linkedmolecular markers were identified. Genetic linkage analysis showed that these markersdistributed in both sides of BcRfp, of which two markers26920and SSR03with the nearestgenetic distances0.44cM and0.7cM, respectively. These markers located the BcRfp gene onchromosome A09with about380kb of Brassica rapa database, which made a basis forfurther positional cloning of the BcRfp gene and for rapid selection of new varieties. |
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