CRISPR/Cas9 Editing Winter Wheat MYC2 Gene And Preliminary Identification Of TaMYC2A,B And D Genes

Low temperature severely limits crop yield and distribution.Jasmonic acid（JA）plays an important role in plant response to low temperature stress and JA biosynthesis is affected by environmental stress.Dongnongdongmai 1（Dn1）is a winter wheat variety with strong cold resistance.Our previous study found that the strong cold resistance of Dn1 was related to JA signal transduction.Exogenous Me JA treatment improved the cold resistance of Dn1.TaMYC2,an important transcription factor in JA signal transduction pathway participates in the cold resistance of winter wheat.MYC2 is the core regulator of JA signaling pathway,which activates the expression of JA responsive genes.In order to analyze the mechanism of JA in winter wheat cold resistance,this study the TaMYC2A,TaMYC2B and TaMYC2D genes of Dn1 were analyzed and predicted by bioinformatics;CRISPR/Cas9 technology was used to edit the TaMYC2 gene of Dn1,the mature embryos were used as explants to establish and optimize the regeneration system,the knock of TaMYC2 gene was obtained by Agrobacterium tumefaciens transformation;The expression levels of TaMYC2 and JA synthesis genes in Dn1 under low temperature stress were determined;The TaMYC2A,TaMYC2B and TaMYC2D genes were overexpressed in Arabidopsis thaliana and the expression changes of cold response genes and JA synthesis genes under low temperature stress were detected to further verify the gene functions of TaMYC2A,TaMYC2B and TaMYC2D.This study will provide a scientific basis for further understanding the hormone regulation mechanism of strong cold resistance in winter wheat and provide new gene resources for genetic improvement of crop stress resistance.The results show that:（1）The full length of TaMYC2A gene is 2088 bp and encodes 693 amino acids.It is located on wheat chromosome 1A 349358465-349360553;The full length of TaMYC2B gene is 2082 bp and encodes 686 amino acids.It is located on wheat 1B chromosome 376071131-376073212;The full length of TaMYC2D gene is 2088 bp,encoding 695 amino acids,which is located on wheat 1D chromosome 277092891-277094978;TaMYC2A,TaMYC2B and TaMYC2D proteins were located in the nucleus,which were irregular curly proteins;The homology of Dn1 TaMYC2A,TaMYC2B and TaMYC2D was 94%,which was close to Arabidopsis At MYC2 and farthest from At MYC146;TaMYC2A,TaMYC2B and TaMYC2D proteins interact with JAZ protein family,COI1 protein,NINJA protein and MPK protein kinase,and At MYC2 protein also interacts with JAZ protein family,COI1 protein and MPK protein,suggesting that At MYC2 and TaMYC2 may play similar roles in JA signal transduction pathway.（2）Three sg RNAs were designed for TaMYC2A,TaMYC2B,TaMYC2D genes,sg RNA1 and3 targeting TaMYC2A,TaMYC2B,TaMYC2D genes,and sg RNA2 targeting TaMYC2B,TaMYC2D genes.The in vitro cleavage efficiency of sg RNA1,2,3 was 90%,70%and 0%,respectively.The TKC vector initiated by Ta U6 was constructed and Agrobacterium tumefaciens was used to infect the callus induced by Dn1 mature embryo.A total of 4406 calli were successfully induced from the mature embryos of 6221 Dn1 seeds with an efficiency of 71%;After Agrobacterium tumefaciens Infection,1014 calli were successfully differentiated with an efficiency of 23%;106 calli were successfully rooted with an efficiency of 10.5%;22 plants were successfully obtained by indoor refining,and the efficiency was 20.7%.The sg RNA expression frame and Cas9 expression frame were detected in 3 of 22 Dn1 TaMYC2 T₀ strains edited by CRISPR/Cas9,which indicated that TKC vector had been successfully integrated into Dn1 genome;There was no phenotypic difference between the three positive plants and the wild type;No mutation was detected in TaMYC2A,TaMYC2B,TaMYC2D genes by T7EI endonuclease,which may be related to the existence of chimera in T₀ generation or the need for deep sequencing.（3）Low temperature stress increased the relative expression of TaMYC2 and JA synthesis genes Ta LOX1,Ta LOX1,Ta LOX1 and Ta OPR2 in Dn1 tillering node,with the highest expression at-10℃;Exogenous Me JA treatment significantly increased 0℃、-10℃and-25℃The relative expression of TaMYC2 in tillering node at 0℃increased the expression of Ta LOX1,Ta LOX2,Ta LOX1,Ta AOS and Ta LOX1.（4）The overexpression vectors of TaMYC2A,TaMYC2B,TaMYC2D were constructed and transformed into Arabidopsis thaliana to obtain T₁ generation positive plants.Overexpression of TaMYC2A,TaMYC2B,TaMYC2D genes significantly increased the relative expression of At COR47,At KIN1 and At LOX6 genes in Arabidopsis plants under low temperature stress.