Screening, Genetic Analysis And Resequencing Analysis Of β-Ocimene-insensitive Mutants

β-ocimene(β-ocimene)is a plant communication signaling molecule that can induce a multi-layered steric defense response in plants,and can relieve the antagonism between the salicylic acid pathway(salicylic acid,SA)related to resistance to living pathogens and the jasmonic acid pathway(jasmonic acid,JA)related to resistance to necrotrophic pathogens and insects,playing a very important role in plant communication and plant defense responses.To identify possible receptor genes for β-ocimene signaling molecules,Arabidopsis thaliana Columbia type seeds were used as materialsin this study,mutagenized and screened by EMS 0.2%(v/v)half-lethal concentration,then stable genetic homozygous mutant lines oims insensitive to β-ocimene were obtained in the M3 generation,mutant lines oim4 and oim7.Have genetic analysis of offspring of oims crossed with wild type;at the same time,the expression of related defense genes PR1 and PDF1.2 was analyzed under the induction ofβ-ocimene,salicylic acid,jasmonic acid,etc;detect the resistance response to biotic stress after induction by β-ocimene.Finally,whole-genome resequencing and bioinformatics analysis of mutant and wild-type materials were performed,and the following findings were obtained:1.A β-ocimene-insensitive mutant was obtained,the mutation was a recessive mutation,and the phenotype was controlled by a pair of alleles.After EMS mutagenesis,35μmol/L concentration of β-ocimene was used for screening(under this condition,wild-type Arabidopsis seeds could not germinate,meaning that wild-type arabidopsis was sensitive toβ-ocimene),and successfully obtained stable inheritance in the M3 generation.The homozygous mutants oim4 and oim7,which are insensitive to β-ocimene,have basically the same phenotype as the wild type.After the mutants were crossed with each other,the seeds of the F1 generation were treated with β-ocimene at a concentration of 35 μmol/L,which all germinated,suggesting that the mutants screened were the same;Seeds did not germinate after β-ocimene treatment,indicating that the mutant trait-related mutation was a recessive mutation,the phenotype of the F2 generation was separated,and the separation ratio of seed ungermination to germination was about 3:1,indicating that the mutant phenotype should controlled by a pair of alleles.2.The relative expression of defense pathway resistance genes was significantly reduced in β-ocimene-insensitive mutants induced by β-ocimene.The mutant and wild type were induced by β-ocimene,jasmonic acid,salicylic acid and ethephon,respectively,and the untreated was used as a blank control.The relative expression levels of PR1,PDF1.2 and VSP2 genes in oim4 and oim7 mutant materials were significantly lower than those in wild-type Col-0.This implies that the gene mutation in the β-ocimene signaling pathway leads to the simultaneous down-regulation of the relative expressions of PR1,PDF1.2 and VSP2 genes in oims mutants.That is,the corresponding gene in the mutant is a component of the β-ocimene signal transduction pathway.3.β-Ocimene induction failed to improve the resistance of mutant materials to biotic stress.Untreated β-ocimene-insensitive mutants and wild-type Arabidopsis materials were used as controls,and oim4 and oim7 treated with 10 μmol/L β-ocimene for 6 hours were used as experimental groups.Pseudomonas syringae,diamondback moth and Spodoptera litura were respectively inoculated to observe their responses to biotic stress.The experimental results showed that the use of β-ocimene could improve the resistance of wild-type Col-0 to Pseudomonas syringae,Plutella xylostella and Spodoptera litura.However,β-ocimene failed to initiate the response of mutants to biotic stress,indicating thatβ-ocimene-insensitive mutants had impaired resistance to biotic stress,and their mutant genes were located in the basilene signaling pathway.4.Four possible candidate genes on the β-ocimene pathway were screened out:AT3G15120,AT5G57720,AT3G28223,AT3G28320.Further whole-genome resequencing and related bioinformatics analysis of mutant and wild-type materials were performed.After excluding the same mutation sites as wild-type,mutant oim4 and oim7 had one identical SNP homozygous mutation site with three identical The homozygous mutation sites of INDEL were located in exons,and the most probable genes for mutation positions were AT3G15120,AT5G57720,AT3G28223,and AT3G28320.