Create A Violaxanthin De-Epoxidase BnaVDE Gene Mutant Of Brassica Napus By CRISPR/Cas9

Sclerotinia rot of colza is a common fungal disease caused by Sclerotinia sclerotiorum in rapeseed planting areas.Presently,the prevention and control of sclerotina disease are mainly chemical and agricultural control.There is a lack of resistant varieties against this disease.S.sclerotiorum invades plant by secreting oxalate thereby acidifying the plant tissue.Furthermore p H in the chloroplast thylakoid lumen decreases,Violaxanthin de-epoxidase（VDE）activity increases,violaxanthin content and ABA accumulation also reduces leading to the opening of the plant stomata.This will lead to decrease in callose and ROS finally resulting in plant sensitivity to S.sclerotiorum.In this study,based on the structural characteristics of BnaVDE,the CRISPR/Cas9 gene editing technology was used to create BnaVDE gene mutant strains to explore the mechanism of interaction between Brassica napus and S.sclerotiorum from BnaVDE.The results of this study are as follows:1)BnaVDE has three conserved domains of the VDEs family.BnaVDE has the amino acid homologous sequence of Bna A06.VDE and Bna C05.VDE.Bioinformatics analysis of Bna A06.VDE and Bna C05.VDE with VDE of Arabidopsis,Rice,and Nicotiana revealed that both Bna A06.VDE and Bna C05.VDE have three conserved domains of the VDEs family:N-terminal cysteine-rich domain,lipid transporter domain and C-terminal glutamate-rich domain.Moreover,the identity of the VDE amino acid sequence is more than 60%,indicating that the VDE amino acid sequences are relatively conserved during evolutionary development.2)Expression of BnaVDE is in all tissues of B.napus.In this research,the expression levels of Bna A06.VDE and Bna C05.VDE in B.napus tissues（root,stem,stem leaf,flower,flower bud,silique,coryledon and rosette leaf）were analyzed.The results showed that Bna A06.VDE and Bna C05.VDE were expressed in various tissues of B.napus,with the highest levels in leaves and varied expression levels among tissues.3)Acquisition of BnaVDE mutant strains.The CRISPR/Cas9 system was used to mutate the BnaVDE by Agrobacterium-mediated genetic transformation.37 strains of Bna A06.VDE transgenic plants were obtained with a positive rate of 63.79%,31 strains of Bna C05.VDE transgenic plants of 83.78%and three Bna A06C05.VDE transgenic plants of10.71%.Among these transgenic plants,yielding 13 strains homozygous for Bna A06.VDE with mutations of 35.14%and 5 strains heterozygous strains and 9 strains homozygous for Bna C05.VDE with mutations of 45.16%.The expression level of Bna A06.VDE was down-regulated in A1 and A46 mutant strains and the expression level of Bna C05.VDE decreased significantly in C20,C25 and C34 plants.4)BnaVDE participates in rapeseed resistance to S.sclerotiorum.Analysis of leaves ofWT plants by q PCR showed that Bna A06.VDE and Bna C05.VDE expression was significantly downregulated.C20 T₀mutant lines together with WT were inoculated with S.sclerotorium.The result shows that there was no significant difference in lesion size between C20 and WT at 12 hpi.However,at 24、36 and 48 hpi,there was significant difference in lesion size between C20 and WT.In summary,BnaVDE has three conserved domains of VDEs family and BnaVDE is involved in Brassica napus resistance to S.sclerotiorum.BnaVDE mutant strain was created by CRISPR/Cas9 technology.This will serve as experimental materials and theoretical basis for the subsequent exploration of BnaVDE function in the process of B.napus against S.sclerotiorum infection.