Construction Of Genetic Transformation System And Gene Editing Of Suts By CRISPR/Cas9 In Neolamarckia Cadamba

N.cadamba is an important fast-growing broadleaf tree speCles with excellent wood properties,and widely distributed in tropical and sub-tropical regions.It can be used as functional materials over a broad range of applications in food,mediClne and feed.With the completion of the whole genome sequenClng of N.cadamba,it is urgent to explore and identify key functional genes involved in xylem development and secondary metabolism,which wo?ld be benefit to functional genomics research and genetic improvement of forest trees in south china.In general,it is critical to establish both the genetic transformation and gene editing systems for the characterization of gene functions.However,little has been reported about on the construction of the two systems in N.cadamba.In this study,high effiClent shoot regeneration from stem and Agrobacterium-mediated genetic transformation system via ?-glucuronidase(GUS)detection were successf?lly constructed.In addition,gene editing of sucrose transporters by CRISPR/Cas9 system was also studied.The main res?lts are as follows:1.By adjusting ratios of different hormones,medium containing MS salts,2 mg/L TDZ,1 mg/L 6-BA,0.1 mg/L 2,4-D and 0.1 mg/L IBA was the best for callus induction when using stems as explant.It was found that 2,4-D and TDZ were the key hormones for callus formation in N.cadamba.The rate of callus induction was 66.67% and the average number of a callus was 93.7.The optimal medium for germination was MS+3 mg/L6-BA+0.3 mg/L NAA whereas MS supplemented with 0.1 mg/L NAA was the most suitable for rooting.2.In order to establish the genetic transformation system of N.cadamba,the effect of explants,hygromyCln concentration,pre-c?lture time,co-c?ltivation time,Agrobacterium concentration,and the time of immersion for transformation were assessed.The res?lts showed that the explants with 4-10 days pre-c?lture were the best for transformation.ThehygromyCln concentration of 16mg/L was suitable for callus induction,but only 10mg/L was optimal for rooting.Co-c?ltivation time and the treatment of explants were supposed to have the greatest impacts on the transformation effiClency in N.cadamba.3.The plasmid pCAMBIA1305 carrying GUS gene was transformed into the stem of N.cadamba by Agrobacterium.The transformants were confirmed by histochemical GUS staining and PCR,revealing that the GUS gene was successf?lly integrated into the genome of N.cadamba.4.CRISPR/Cas9 system was used for gene editing of NcSUT2,NcSUT3 and NcSUT4 in N.cadamba.Five constructs with three-target sites were generated,including pYL CRISPR/Cas9-gRNA-SUT2,pYLCRISPR/Cas9-gRNA-SUT3,pYLCRISPR/Cas9-gRNASUT4-1,and pYL CRISPR/Cas9-gRNA-SUT4-2 and pYL CRISPR/Cas9-gRNA-NCSUT.They were transformed into the stem of N.cadamba by using the genetic transformation system established in our study.A total of 36 transgenic plants were obtained and verified by PCR and sequenClng,of which 12 plants were characterized with off-target SUT DNA cleavage,indicating high rate of off-target DNA cleavage by CRISPR/Cas9 system under35 S promoter in the woody speCles N.cadamba.In summary,the genetic transformation systems of GUS gene and CRISPR/Cas9 were successf?lly constructed based on the system of shoot regeneration from stem in N.cadamba.Furthermore,preliminary study on gene editing of SUTs genes was performed.These res?lts laied foundation for the functional genomics research in N.cadamba in the future.