Cloning Of MtCOMT Gene,Construction Of CRISPR/Cas9 Vector And Transformation Of Medicago Truncatula

Lignin is a kind of complex aromatic polymer,which plays a mechanical supporting role in the process of plant growth and development.In addition to absorbing soluble proteins in alfalfa,herbivorous livestock can also decompose and absorb polysaccharides such as cellulose and hemicellulose,which constitute cell wall components.However,due to the presence of lignin in the cell wall,the digestion and absorption of these polysaccharides in the rumen of herbivores are seriously hindered.Caffeic acid O-methyltransfease(COMT)is a neighboring methyltransferase,which plays an important role in the biosynthesis of lignin unit and determines the synthesis of syringyl(S)lignin unit.Therefore it can reduce the content of lignin by inhibiting COMT content in Medicago truncatula.As the third generation genome editing technology,CRISPR/Cas9 system is a reliable,simple and efficient genome editing tool.It is able to manipulate crop genomes through genetic modification to produce new varieties which resemble natural mutations.It plays an important role in the crop genome editing.Based on the above reasons,in our study,MtCOMT was cloned from R108 of Medicago truncatula.Using CRISPR/Cas9 genomic editing platform based on hairy root culture system,MtCOMT gene in R108 was edited at a fixed point to obtain edited homozygous hairy root system.And the genetic resources of alfalfa with Mt COMT mutation were produced,that laid the molecular foundation for genetic to improve the quality of alfalfa.The main results are as follows:1、Cloning and sequence analysis of MtCOMT geneThe electronic sequence of MtCOMT(gene number is Medtr3g092900)in Medicago truncatula was obtained by phytozome public resource database and we designed primers to clone the Mt COMT.The sequence of MtCOMT contains 1098 nucleotides,which can encode 365 amino acids.Its relative molecular weight is39.921 kda,and the theoretical isoelectric point is 5.67.NCBI protein blast analysis shows that the protein had a dimerization domain in 34-85 amino acids and a methyltransf 2 conservative domain in 140-345 amino acids,which has theS-adenosylmethionine binding site.Furthermore,MtCOMT and MsCOMT are closely related with 99.73% homology.There is only one amino acid mutation in the conserved domain,and Ser was replaced by Leu at 80 th amino acid.2、Construction of CRISPR/Cas9 gene editing vectorAccording to the electronic sequence of MtCOMT,two sgRNA targets were designed on the first and second exons of MtCOMT.On the basis of pYLCRISPR/Cas9-P35 S skeleton vector,the promoter AtU3 b and BsaI enzyme were connected.And two tRNAs conserved sequences were introduced to separate two 20 nt sgRNAs.The constructed vector contained sgRNAs and Cas9 expression cassette at the same time.The plant expression vector pYLCRISPR/Cas9-AtU3b-tRNA-COMT was successfully constructed.3、Selection of editing homozygous hairy rootThe pYLCRISPR/Cas9-AtU3b-t RNA-COMT plant expression vector was transformed into Medicago truncatula by Agrobacterium rhizogenes mediated leaf disc transformation.The DNA of hairy roots was extracted by 2×CTAB method,and identified by PCR with Cas9 primer,Bar primer,RolB primer and MtCOMT primer.The positive clones were identified by BsaI enzyme.Three hairy root systems were identified by PCR/RE.Through further sequencing analysis,the results showed that none of the three root systems had mutation at the first target and 100% mutation at the second target,which further proved that the three hairy root systems were editing homozygous.4、Propagation and regeneration of the editing homozygous hairy rootThe homozygous hairy root systems were cultured continuously,and the single hairy root systems were expanded by induction medium to prepare experimental materials for further study.At the same time,the edited homozygous hairy roots were transferred to the medium.After callus induction,differentiation regeneration and rooting,the regenerated plants of MtCOMT were obtained.