Molecular Mechanism Of Silique Enlargeent Based On A Super-Long Silique Mutant In Brassica Napus L.

Rapeseed is an important oil crop in China and the largest source of edible vegetable oil in China.A super-long silique mutant Bnspt was obtained in our previous study,which showed the phenotype of silique enlargement.In this study,the molecular mechanism of silique enlargement was studied based on Bnspt mutant.The main results are as follows :(1)To preliminarily investigate the molecular mechanism of silique enlargement in the super-long silique mutant Bnspt,the silique of Bnspt and CK were selected for RNA-seq analysis.The results showed that there were 2999 differentially expressed genes,including 2109 up-regulated genes and 890 down-regulated genes.GO enrichment results showed that the largest number of differentially expressed genes was in the ‘ Biological Process ’ category;the KEGG enrichment pathway showed that the two pathways with the largest number of differentially expressed genes were Metabolic pathways and Biosynthesis of secondary molecules.(2)Further,we selected the differentially expressed genes in plant hormone signaling pathways for q-PCR analysis.The results showed that the expression levels of the tested genes in auxin,cytokinin,brassinolide,abscisic acid and gibberellinmediated pathways were significantly changed.It was suggested that the signal transduction pathways of these hormones in Bnspt played a role in the regulation of silique size in B.napus.(3)Further,we found a gene Bn CPD containing PIN domain from transcriptome differentially expressed genes.PIN domain exists in the animal ZC3H12A/B/C/D protein family,which is related to cell proliferation.In order to study whether Bn CPD has the function of regulating silique size,we successfully cloned this gene from B.napus.Furthermore,we constructed Bn CPD overexpression vector and transformed it into Arabidopsis thaliana.The results showed that the main roots and siliques of Bn CPD overexpression plants were significantly increased and enlarged,and had the phenotype of early flowering.(4)To further explore the function of this gene,we established Bn CPD overexpression and RNAi suppression transgenic plants.The results showed that the over-expression plants showed the phenotype of plant enlargement,early flowering,silique enlargement and seed enlargement,which was consistent with the results in Arabidopsis thaliana.Furthermore,q PCR analysis showed that the expression levels of auxin,brassinolide,abscisic acid and gibberellin-related genes in the test were significantly changed,suggesting that the signaling pathways of these hormones in Bn CPD overexpression plants may be involved in the regulation of silique size.The results in Arabidopsis and B.napus showed that Bn CPD plays an important role in regulating plant silique size.Then,we investigated the phenotype of Bn CPD RNAi transgenic plants.The results showed that RNAi plants showed a shorter phenotype than CK,because of the time relationship,we will do further research on RNAi transgenic lines.(5)In addition,we selected another candidate gene Bn CBS from transcriptome differentially expressed genes.Knockout of Bn CBS homologous genes in Arabidopsis showed insufficient accumulation of reactive oxygen species(ROS)and smaller siliques.In order to study whether Bn CBS has the function of regulating the size of silique,we cloned this gene from B.napus and constructed the overexpression vector of Bn CBS.The results showed that the length of the main root and silique of the overexpression plants did not change significantly.DAB staining showed that Bn CBS overexpression significantly increased ROS accumulation,indicating that Bn CBS may be involved in redox balance in plants.Further,we created Bn CBS over-expression and RNAi transgenic rape plants.However,due to the delay of experiment,further research will be done in the future.