Possible Roles Of Several Soybean Domestication-related Genes In Response To Abiotic Stress

To clarify the potential roles of soybean domestication-related genes in responses to abiotic stresses,we selected seven soybean domestication-related genes,GmRACK1 C,GmFBA6,GmbHLH,GmZFP,GmChiC,NAC4 and NAC27,and systematically studied the alterations of their expression levels following abiotic stress treatments,such as drought,hypersaline and ABA,based on bioinformatics analysis of the cis-regulatory elements upstream from the promoter regions by qRT-PCR.Knockout mutants of these soybean domestication-related genes and their homologs from Arabidopsis thaliana were also generated using CRISPR/Cas9 transgenic technology and the differences between the responses of various mutants and wild types to different abiotic stresses were then compared.Our main results are as following:1)Cis-acting elements related to hormones,abiotic stresses and other regulations were detected in every promoter region of the seven soybean domestication-related genes,indicating that their expression are probably regulated by abiotic stresses.The qRT-PCR analysis showed that five genes including GmbHLH,GmZFP,GmChiC,NAC4 and NAC27 were sensitive to abiotic stresses.Among them,GmbHLH and GmZFP were significantly down-regulated in response to drought and high salinity,while GmChiC,NAC4 and NAC27 were significantly up-regulate to the same stresses.2)Genes from Arabidopsis are of the greatest similarities from their soybean domasetication-related homologs.We successfully obtained knockout lines of four Arabidopsis homologs including RACK1 C,FBA6,bHLH and ANAC055 using CRISPR/cas9 transgenic technology.Phenotypic trait measurements and stress resistance analysis showed that compared to those in wild type plants,both the length of the root and the shoot fresh weights of bHLH knockout mutants were significantly increased,and the reduction of shoot fresh weights were significantly decreased in bHLH mutants following drought treatment;However,the shoot fresh weights ofANAC055 knockout mutants were significantly reduced,and the reduction of germination rates and shoot fresh weights were significantly decreased to drought and high salt stress in knockout lines than in wild types.The results indicated that bHLH and ANAC055 may play important roles in arabidopsis abiotic stress responses,such as drought,with varied mode and mechanism of action may be different.3)Using the CRISPR/Cas9 transgenic technique,soybean GmRACK1 C,GmbHLH,GmZFP,GmChiC and NAC4 knockout lines were successfully obtained.All mutations in four target genes(GmRACK1C,GmbHLH,GmZFP,GmChiC)are shift mutations leading to alterations in the amino acid sequences of the coding proteins,and the mutation of NAC4 resulted in one amino acid loss in its encoded protein.These mutants provide good materials for further researches on the role of soybean domestication related genes in abiotic stress.Our study focuses on the possible role of soybean domestication-related genes in abiotic stress resistance,which is of great significance to reveal the molecular process of soybean domestication and the genetic basis and molecular mechanisms of soybean resistance to abiotic stress.