Study On CRISPR/Cas9 Editing Of CCoAOMT And ALS Genes In Medicago Sativa

Alfalfa(Medicago sativa)is a high-quality forage widely cultivated all over the world and occupies an important position in national economy.High lignin content and weeds are the key factors restricting the sustainable development of alfalfa industry with high yield and high quality.At present,commercial alfalfa varieties with low lignin and herbicide resistance are very rare,and precise molecular design breeding is helpful to create new alfalfa germplasms quickly and effectively.In this study,the members of MsCCoAOMT gene family related to lignin biosynthesis and MsALS gene family related to herbicide were identified from genome wide for the first time,and their gene expression was analyzed.Using CRISPR/Cas9 technology to edit MsCCoAOMT and MsALS,the gene editing system of alfalfa was preliminarily established,and the target gene edited materials were obtained.It laid a foundation for breeding new alfalfa materials of reducing lignin content and herbicide-resistant.The main research results are as follows:1.The MsCCoAOMT gene family was identified from genome wide level.A total of 44 MsCCoAOMTs genes were identified.Phylogenetic tree analysis showed that they distributed in 5 evolutionary branches,and had high similarity with CCoAOMT of Medicago truncatula,Arabidopsis thaliana and rice.The q RT-PCR analysis showed that 11 MsCCoAOMTs expressed tissue specifically in stems and leaves of alfalfa during seedlings and vegetative stage,and stems,leaves and flowers during flowering stage.2.The MsALS gene family was identified from genome wide level.A total of 31 MsALS genes were identified and distributed on 19 chromosomes of alfalfa,including6 pairs of tandem repeat genes.Except for MsALS23,MsALS29,MsALS30 and MsALS31,all MsALS proteins have three typical conserved domains: TPP enzyme N,TPP enzyme M and TPP enzyme C.Based on phylogenetic tree,MsALS2,MsALS5,MsALS9,MsALS12 and MsALS13 are closely related to At ALS(AT3g48560.1).qRTPCR analysis showed that MsALS5 had the highest relative expression in the stems at the flowering stage,and MsALS12 had the highest relative expression in the leaves at the seedling stage.3.Mutant alfalfa plants of MsCCoAOMT gene-editing were obtained.Two sg RNAs were designed in the conserved region of the MsCCoAOMT,and the CRISPR/Cas9 expression vector,simultaneously knocked out 6 MsCCoAOMTs(MsCCoAOMT6/12/13/14/15/18)genes was constructed.A total of 427 transgenic alfalfa plants were obtained,with a positive rate of 70.7%.Three mutants with base insertion at the target site were screened.4.MsALS gene-edited transgenic alfalfa plants were obtained.Five MsALSs(MsALS2/5/9/12/13)genes were edited by CRISPR/Cas9 technology,and positive transgenic alfalfas were obtained.The herbicide resistant seedlings were identified by sequencing.The effective concentration of nicosulfuron herbicide at alfalfa seedling stage was 600 mg/L and imazapic herbicide was 1350 mg/L by a concentration gradient screening.The screening of herbicide-resistance on mutant plants has not yet been completed.