Construction Of CRISPR/Cas9 Genome Editing System And Its Editing Effect On BnSVP In Rapeseed (Brassica Napus L.)

Rapeseed,an annual or biennial herb belongs to Brassica,Cruciferae,has three cultivated types as Brassica rapa?AA,2n=20?,Brassica juncea?AABB,2n=36?and Brassica napus?AACC,2n=38?.Rapeseed is one of the four major oil crops in China and the third largest ecocomic crop in the world.Brassica napus has been the main culitiviation type across the major oilseed production regions in China because of its good disease-resistant and insect-resistant performance and high yields.CRISPR/Cas9system is emerging as a new geome editing technology for molecular modification,by which the target gene will change or lose functions because of deletion,insertion or substitution of several nucleotides in specific sites,so it is a powerful tool for deciphering biological functions of genes.In this study,an elite and model cultivar of Brassica napus?Zhongshuang No.11?was used as an experimental material.We explored several factors affecting regeneration efficiency using its hypotocls and cotyledonary petioles as explants,and constructed several genome editing expression vectors targeting homology SHORT VEGETATIVE PHASE?SVP?in Zhongshuang No.11 genome based on CRISPR/Cas9system.To evaluate the editing efficiency of this genome editing system,two of six recombinant vectors were selected and studied using protoplast transient expression system.The main results are as follows:1.To establish an efficient regeneration system for genome editing system using cultivar Zhongshuang No.11,factors affecting callus induction and differention like carbon source,AgNO₃,hormone combinations and concentrations and callus inducing periods were investigated using hypotocls and cotyledonary petioles.Results showed that 5-day-old hypotocls had a 55%shoot regeneration ratio culturing on callus induction medium sumpplemented with 30g/L glucose and l.0 mg/L 2,4-D for 7 days then followed culturing on differential medium sumpplemented with 3.0 mg/L6-BA and2.5 mg/LAgNO₃,while 5-day-old cotyledonary petioles had a 70%shoot regeneration ratioculturing on callus induction medium sumpplemented with 30g/L sucrose and l.5mg/L 2,4-D for 7 days then followed culturing on differential medium sumpplemented with 4.0 mg/L6-BA and 2.5 mg/LAgNO₃.2.Four SVP homologs were isolated by homology searches against Zhongshuang No.11 genome.On the basis of alighments and sequence analyes,twelve sgRNA seed sequences targeting four homologous copies of SVP in Zhongshuang No.11 were designed by using software CRISPR-P 2.0,and then six dual sgRNAs CRISPR/Cas9genome editing constructs were developed employing pYLCRISPR-Cas9P35S-H as backbone and AtU3b or AtU3d as promoter with Golden Gate Cloning technology.Sequencing results demonstrated that all the recombinant plasmids showed the correct nucleotides and expression cassette assembly.3.To evaluate the editing efficiency of this genome editing system,two vecotors simultaneously targeting four copies of BnSVP were seleced and transfected by protoplast transient expression assay.Specific PCR products of BnSVP in A4?A9 and C4 linkage groups were harvested applying nested PCR technique.Sequencing results demonstrated that only a transition?A?G?happened at the 4^th base upstream of the PAM site of T3 taget within BnSVP.A4,which resulted in a transition from Asp?D?to Gly?G?,and the average editing efficiency was 33.33%.