Genome Editing Of BnMlO6 By CRISPR/Cas9 To Improve Disease Resistance In Brassica Napus L.

Brassica napus is an important oil crop.Fungus diseases cause heavy yield loss in B.napus.CRISPR/cas9 system can achieve accurate editing of target genes,opens up a new way for improving plant disease resistance.Mildew resistance locus O（MLO）gene is a key negative regulator of plant defense against powdery mildew.Mutation of MLO gene could enhance plant resistance to powdery mildew,but whether it has the same function in B.napus has not been reported.In this study,we analyzed the expression of Bn MLO gene after inoculation with Sclerotinia sclerotiorum in B.napus.We used CRISPR/cas9 technology to knock out Bn MLO6 gene,and analyzed the effect of mutantion on the resistance powdery mildew and S.sclerotiorum.The results are as following:1.Based on the amino acid sequence of Arabidopsis At MLO protein and sequence similarity analysis,we obtained the homologous genes of MLO2 and MLO6 in B.napus from the reference genome Darmor-bzh,and found that there were four homologous of Bn MLO2 and six homologous of Bn MLO6 gene in B.napus.After inoculation with S.sclerotiorum,the expression of Bn MLO genes in B.napus was analyzed.The expression of Bn MLO6 increased significantly and reached the peak 24hours after inoculation,while the expression of Bn MLO2 decreased significantly alone inoculation time.It inferred that expression of Bn MLO6 gene was induced by S.sclerotiorum,.2.In order to construct a binary vector,sg RNAs were designed to target two common conserved sites of six homologous of Bn MLO6.We introduced the editing vector into“Zhongshuang 6”by Agrobacterium mediated transformation.In this experiment,494 regenerated plantlet were obtained,and 137 plants were positive,with a positive rate of 27.7%.After screening the differential bands by PAGE electrophoresis,41 edited plants were obtained.The mutation efficiency of p KSE401-Bn MLO6vector was 29.9%.Among 41 edited plants,ten plants with more than one Bn MLO6 homologous mutated and one coded mlo6-212 was identified with six Bn MLO6 homologous mutated.The seeds harvested from mlo6-212 were grew in Hanchuan transgenic experimental station.All mutation types of T₀generation were detected in T₁generation without any new mutation type.In the T1 plants derived from mlo6-212,segregation of mutation types were observed,but each possessed at least four homologous mutation,indicating that the mutation produced by CRISPR/Cas9 system could be inherited stably.3.In Hanchuan experiment station,almost all wild type rapeseed lines were infected with powdery mildew at mature stage,but Bnmlo6 mutants was not infected.It indicated that the mutants had strong resistance to powdery mildew.On the other hand,S.sclerotiorum infection was performed with wild type and plants of mlo6-212 T₂generation.Compared with non-transgenic lines,the lesion areas on the detached leaves of mlo6-212 T₂plants were significantly decreased.Compared with the wild type,the lesion area of Bnmlo6 mutant decreased by 19.5%（P=0.012）at 24 hpi,strongly suggesting that Bn MLO6 mutation increased the resistance to S.sclerotiorum in rapeseed.We observed callose in T₁mutant by fluorescent microscopes and found that the number of callose dots in T₁mutant increased significantly under natural conditions.The increase of callose may be one of the important factors to enhance the disease resistance of mutants.The expression of jasmonic acid（JA）and ethylene（ET）pathway marker genes in mutants and wild type plants were analyzed after inoculation.The results showed that the expression of ERF1,ERF2,PR4 and ORA59 genes in the mutants was earlier and higher than that in the wild type.This suggests that Bnmlo6 may mediate the JA/ET signaling pathway and increase the resistance to S.sclerotiorum.