| Brassica napus is one of the important oil crops in the world.China is a major consumer of vegetable oil,a large number of vegetable oil is still dependent on imports.65% of the total oil consumption needs to be imported,so it is imperative to increase rapeseed production to reduce vegetable oil imports.Therefore,it is important to explore the mechanism of the branching development of rapeseed,which can affect the yield of rape.In this study,the BnSFZ gene was cloned from mutant sfz,and the function of the gene was analyzed,so as to enrich the understanding of the molecular mechanism of the branch development of rape.The following is the main results of this study:1.Phenotypic analysis of mutant sfzOur laboratory has successfully obtained mutant sfz containing the activated tag vector PXT246.The assay of the sfz phenotype showed that the effective branch number decreased and height of primary branches increased in the mature period.The content of fatty acids in rape sfz seeds was not significantly different from that of wild type plants by near infrared spectroscopy.In the infection experiment of Sclerotinia sclerotiorum,the leaf of the mutant sfz was infected quickly and the plaque area was larger than NY12.2.Protein differential expression analysis of mutant sfz16 differentially expressed protein spots was isolated and identified by 2-DE technique from the total protein of NY12 and the mutant sfz leaves;Under the analysis of mass spectrometric and database search,we matched the candidate protein.These proteins are closely related to the material and energy metabolism,stress,resistance,energy generation,growth development,signal transduction and physiological activities,they also play important function and role in rapeseed growth and development.3.Flanking sequence amplification and analysis of mutant sfz.hiTAIL-PCR and FPNI-PCR have been applied to amplify flanking sequence of mutant sfz.The flanking sequence was cloned by FPNI-PCR.The genes SFZ1,SFZ2,SFZ3 and SFZ4 near the insertion site were obtained by software analysis.4.Expression and bioinformatics analysis of candidate genesqRT-PCR analysis showed that the expression of SFZ4 gene was increased in mutant sfz plants,and there was no change in the rest.SFZ4 was used as the final candidate gene and named BnSFZ.Bioinformatics analysis revealed that BnSFZ protein belongs to AP2 family,and plays an important role in plant growth and development.5.Candidate gene complementation assayIn order to further verify the candidate gene BnSFZ,we constructed the over expression and inhibitory expression vector of BnSFZ,and transformed it into wild type rape by Agrobacterium mediated transformation.6.GWAS analysisBased on the results of whole genome association analysis of rape branch number,the SNPs and Indel loci were detected in the 20 Kb sequence before and after the insertion site,and the correlation between these loci and plant branching traits was analyzed.The results showed that SFZ-SNP-18409641 locus had significant effect on branching traits(P<0.01),The site is located in the promoter region of the BnSFZ gene.The results indicated that the BnSFZ gene was correlated with the number of branches in natural population association analysisThe results of this study showed that the expression of BnSFZ gene in the mutant sfz was increased,resulting in the decrease of plant branches and wax content.The development of this study is helpful to understand the molecular mechanism of plant branching and wax synthesis. |
