Functional Identification Of MiR164 In Soybean Responses To Salt Stress

Soybean(Glycine max(L.)Merr.),as an important grain and forage crop in the world,plays a key role in human’s lives because that it contains rich nutritional resources.Soil salinization is a worldwide ecological problem that threats crop production.Although soybean is a moderately salt-tolerant crop,salt stress severely affects quality and yield of soybean.Therefore,exploring the mechanism of salt response in soybean for breeding elite salt-tolerant soybean variety,has great significances for increase of grain yield,the rational use of marginal land,and the effective improvement on the ecological environment.Micro RNAs(miRNAs)are a class of small single-stranded non-coding RNAs,which play the key role in the regulation of plant responses to salt stress.Through a series of genetic and molecular biological methods,we have identified three miRNAs including miR164 d,miR164 g and miR164 k,which participate in the process of salt stress response in soybean.Their biological functions were analyzed,and the detailed research results are as follows:1.Transgenic hairy roots with reduced expressions of miR164 d,miR164 g and miR164 k were achieved through CRISPR/d Cas9 system.By salt treatment on transgenic hairy roots,relative root grown rates of transgenic hairy roots were only 0.16～ 0.35 times of control.In addition,relative root growth rates of both miR164 g and miR164 k overexpressing hairy roots were 1.3 and 1.5 times of control,respectively.These results indicate that miR164 d,miR164 g and miR164 k positively regulate salt response in soybean.2.Via the analysis of expression patterns of miR164 d,miR164 g and miR164 k under salt treatment,we found that their expressions were induced by salt treatment.Their expression levels reached the highest under salt treatment at 6 h,was 6 ～ 20 times of control.Through analyzing expression patterns of miR164 d,miR164 g and miR164 k from different soybean tissues,we found that they expressed in roots,stems,leaves and flowers,and highly expressed in leaves.3.For creating miR164 d,miR164 g and miR164 k knockout mutants through CRISPR/Cas9 system,transgenic plants containing CRISPR knockout vectors were produced by soybean genetic transformation.For miR164 d,two independent homozygous mutated lines were achieved,and their expression levels were only 0.3and 0.25 times that of the wild type,respectively.For miR164 g,one homozygous mutated line was generated.For miR164 k,two independent homozygous mutated lines were created.In conclusion,our study found that miR164 d,miR164 g and miR164 k positively regulate salt response in soybean,which provides both theoretical bases and genetic resources for breeding elite salt-tolerant soybean varieties.