Research On Cl~-/salt Tolerance And Physiological Mechanism Of Soybean GmCLC-c1/2

As one of the most common oil crops in the world,soybeans have high protein and oil content and play an important role in the human diet.As major abiotic stress,salt damage inhibits soybean yield and quality.This work takes cultivated soybean Lee68 as the research object,searches and analyzes soybean CLCs family through soybean database and bioinformatics software,using RNA interference,soybean root transformation,quantitative PCR,tobacco transient expression technology,yeast supplement and the genetic transformation of Arabidopsis technology explores the responsiveness of GmCLC-c1/2 to salt stress.The main results are as follows:Analysis of the gene structure and conserved domains of the soybean CLCs family showed that GmCLC1,GmCLC-b1/2,GmCLC-c1/2,GmCLC-d1/2,and GmCLC-g are all of the same subfamily,and the conserved gene sequences of each member are similar.At the same time,five types of hairy root-composite plants,GmCLC1-RNAi,GmCLCb-RNAi,GmCLCc-RNAi,GmCLCd-RNAi,and GmCLCg-RNAi,were constructed.Through the observation of phenotype and ion content,we found that salt GmCLCc-RNAi plants were the most injured under stress.Cl^-and Na⁺content increased significantly in stems and leaves,while NO₃^-and K⁺content decreased significantly in roots,stems,and leaves.Under salt treatment,GUS expression increased in tobacco leaves injected with Agrobacterium suspension containing Pro GmCLC-c1::GUS and Pro GmCLC-c2::GUS.Besides,through quantitative PCR technology,we found that GmCLC-c1 and GmCLC-c2 are expressed in soybean roots,stems,leaves,flowers,pods,and seeds.At the same time,the expression levels of GmCLC-c1 and GmCLC-c2 in soybean roots and leaves were significantly increased after Na Cl treatment,especially in the roots.On this basis,GmCLC-c1-OE and GmCLC-c2-OE soybean hairy root-composite plants were constructed.Under salt stress,the degree of salt damage to GmCLC-c1-OE and GmCLC-c2-OE soybean hairy root-composite plants is reduced.It maintains the ion balance in the soybean body and reduces the Cl^-and Na⁺content in the stem and leaves.And it increases the content of NO₃^-and K⁺in roots stems and leaves.We transferred GmCLC-c1and GmCLC-c2 into the yeastΔgef1 mutant and found that both of them can restore the growth ability ofΔgef1 under salt stress.Through systematic evolutionary relationship analysis,it found that Arabidopsis At CLC-c is closely related to GmCLC-c1 and GmCLC-c2.Therefore,the Arabidopsis atclc-c mutant was selected as the experimental material,and GmCLC-c1 and GmCLC-c2 were transformed into it.Under salt stress,the germination rate and root length of transgenic Arabidopsis were significantly higher than that of mutants.At the same time,the fresh weight,dry weight,relative water content,and relative electrical conductivity reflected that GmCLC-c1 and GmCLC-c2 complementing plants were affected.The degree of damage is equivalent to that of the wild type.At the same time,it was observed that GmCLC-c1 and GmCLC-c2 can reduce the accumulation of Cl^-in the shoots of Arabidopsis,improve the storage capacity of NO₃^-,and synergistically affect the distribution of Na⁺and K⁺.Also,the content of hydrogen peroxide and superoxide anion in transgenic Arabidopsis was reduced.In summary,this study found that the expressions and the promoter activities of GmCLC-c1 and GmCLC-c2 can be up-regulated by salt induction.Whether in the soybean root-composite hairy plants or transgenic Arabidopsis,GmCLC-c1 and GmCLC-c2 can reduce the accumulation of Cl^-in the shoot of the plant,increase the storage capacity of NO₃^-,and synergistically affect the distribution of Na⁺and K⁺,thereby they maintain the ion homeostasis of the plant under salt stress.However,there is no significant difference between GmCLC-c1 and GmCLC-c2 in ion transport activity.