Study On IDA In Regulating Floral Organ Abscission And Pod Shatering In Brassica Napus L.

Brassica napus L.is the main oil crop in China,providing nearly half of Chinese vegetable oil.The infection of Sclerotinia sclerotiorum and pod cracking are the main factors causing rapeseed yield loss.In addition,B.napus is also used for viewing.The extension of flowering period can greatly enhance its economic value.In this study,the homologous gene INFLORESCENCE DEFICIENT IN ABSCISSION(IDA)related to the non-abscission of floral organs in Arabidopsis was cloned from B.napus,and the gene was named BnaIDA.Bioinformatics analysis showed that BnaIDA was a hydrophobic protein;the phylogenetic relationship of the phylogenetic tree showed that the BnaIDA gene on chromosomes A07 and C06 was the closest to the At IDA gene of Arabidopsis.Furthermore,the tissue-specific expression pattern was analyzed by transcriptome data and qRT-PCR experiment.It was found that the expression level was relatively increase in flowers and mature pods,indicating that BnaIDA probably involved in the process of organ abscission.The subcellular localization of onion bulb inner epidermis showed that BnaIDA-A07/C06 protein was located in the intercellular space,which was consistent with the characteristics of secretory protein and confirmed the results of bioinformatics analysis.In this study,the gene editing vector of site-specific knockout BnaIDA-A07/C06 was constructed,and the positive strain with effective editing at the target and non-shedding phenotype of flower organs was successfully obtained by using rapeseed hypocotyl transformation method.It was found that the phenotype could still be inherited stably in the T2 generation without Cas9 sequence produced by selfing.In order to improve the efficiency of subsequent identification,we designed rapid identification primers according to the editing frequency and the principle of primer 3’ end specific amplification.Mutants genotype can be quickly identified by PCR,which has been successfully verified in the T2 generation of positive strains and contribute to accelerating the breeding process in the future.In addition,compared with WT and mutants,the floral organs of overexpression positive plants shed very quickly,which indicates that BnaIDA-A07/C06 gene plays a regulatory role in the shedding of floral organs in B.napus.We continued to test the dehiscence of mature siliques of mutants and WT,and found that the dehiscence ability of mutants increased significantly,that is,the crack resistance is enhanced.Finally,the disease avoidance phenotype of mutant floral organs not falling off against S.sclerotiorum was tested in the incubator environment.The results showed that compared with the WT,the mutant flower organs did not fall off and the mycelial growth rate of S.sclerotiorum slowed down,but the plants could still grow normally,which greatly reduced the loss caused by the infection of S.sclerotiorum.Subsequently,the RNA-Seq analysis of Bnaida and WT showed that after the deletion of IDA protein,the gene expression levels of receptor proteins HAE and HSL2,MAPK6 in signal cascade amplification pathway and transcription factors BP/KNAT1 and KANT2 increased significantly.But the expression of CEL1 gene encoding cell wall degrading enzyme decreased significantly,which probably affect the separation of cells in the abscission zone,and eventually lead to the phenotype that flower organs do not fall off and the silique dehiscence-resistance was enhanced phenotype.This project successfully uses CRISPR/Cas9 technology to edit in plants,which greatly shortens the acquisition cycle of new germplasm.We provide new germplasm that is more suitable for breeding,with petals non-shedding and the silique dehiscenceresistance was enhanced.It also provides a new breeding material for mechanization and avoiding S.sclerotiorum.