| Soil salinization has been a global ecological problem for a long time,which had an extremely unfavorable impact on agricultural production.In many areas of our country,soil salinization has become the main reason for restricting the growth and development of plants,and it is also the main reason for crop yield reduction.The soybean is a traditional edible leguminous crop that can fix nitrogen among the few plants.Due to the nitrogen fixation capacity,it can significantly reduce nitrogen use as compared with most crops and thus it plays an extremely important role in improving the soil and the atmospheric environment.Therefore,our exploration of soybean salt tolerance can improve the ability of soybeans to adapt to adverse environments.Soybean salt tolerance is a complex trait controlled by multiple genes.At present,the research on the mechanism of soybean salt tolerance is mainly focused on reconstructing ion balance in cell,maintaining osmotic balance,repairing oxidative balance and other metabolic or structural adjustment.Through the excavation of salt-tolerant genes(such as SOS1 and NHX1,the salt tolerance of the corresponding transgenic plants has been improved,but there is no significant improvement in stable and high yield of transgenic salt resistant varieties.Therefore,to improve the salt tolerance of soybean,it is necessary to study the molecular mechanism of soybean salt stress deeply.It is important to improve soybean yield by exploring the mechanism of soybean salt tolerance,improving soybean tolerance,better utilization and improving stress environment(such as saline soil).In adverse circumstances,soybean would suffer variety of stress,such as ion stress,drought stress,oxidative stress,etc.Among them,ion stress is a very important stress factor for soybean.In our study,Soybean N23674(G max)and BB52(G soja)were used as the experimental materials and the GmCLCs genes of soybean were completely cloned and analysized.Furthermore,the GmCLCl and GmCLC-c2 genes of soybean had been explored deeply which have obvious alleviation effect on salt stress.We downloaded the sequences of GmCLCs genes from public network database NCBI(https://www.ncbi.nlm.nih.gov/)and the Pfam database(https://pfam.xfam.org/)through the conservative domain analysis and homologous alignment based on the amino acid sequence of the GmCLCl gene.Besides,we performed bioinformatics analysis on GmCLCs gene of soybean by the bioinformatics software such as Clustalx,Genedoc,MEGA 6.0 and MapInspect,etc.The results show that the GmCLCs family of soybean consists of 8 copies,distributed on chromosomes 1,5,9,11,13,16 and 19,which containing 4 to 23 introns and 725 amino acids 823.The overall similarity is 63.43%and the theoretical isoelectric point ranges from 6.45 to 8.94,with a positive or negative charge amino acid residues ratio of 7%to 10%.They are all transmembrane proteins(containing 9 to 12 transmembrane regions),which are close to the CLC protein of Arabidopsis subfamily I.The soybean cotyledons or cotyledon hypocotyl were injected by Agrobacterium tumefaciens K5 99 to induced root hair and formed soybean hair root composite plants which containing GmCLCs gene.The relative electrolyte leakage rate,chlorophyll content and wilt rate of soybean roots and leaves were determined under 120 mM NaCl stress for 15 days and then the response of GmCLCs gene to NaCl stress was analyzed and compared.The results showed after overexpression of GmCLC-bl or GmCLC-c2 in the roots of soybean,the growth phenotype of soybean plants was significantly improved compared with the control under 120 mM NaCl stress.The relative electrolyte leakage rate,chlorophyll content and wilt rate of the plants reached significant differences.However,when the GmCLC-dl gene was overexpressed at the root of soybean,the growth phenotype of soybean seedlings became worse than that of the control.For example,the relative electrolyte leakage rate and the wilt rate of the plants are significantly increased.And at the same time,the chlorophyll content is significantly decreased.While the overexpression of other members of the GmCLCs genes the growth phenotype of soybean plants also were improved,but none is significant than the transgenic plants which were overexpressed GmCLC-bl or GmCLC-c2 genes.Through the quantitative real-time PCR,we studied the expression of GmCLCs in the roots,stems and leaves of soybeans and the mechanism of response to salt stress.The qRT-PCR result analysis showed that the expression of GmCLCs gene decreased first and then increased,in the process of 150 mmol·L-1 NaCl stress for 24 h.The responses of GmCLC1,GmCLC-b1,GmCLC-b2,GmCLC-c1 and GmCLC-c2 to NaCl are more significant in roots and stems,whereas GmCLC-b1,GmCLC-b2 and GmCLCl are up-regulated more significantly in leaves.In addition,we also introduced the GmCLCs genes into yeast mutants(gefl)which deleted the chloride ion channel gene(GEF1)of yeast We found that the function of the GEF1 gene was well complementary and the growth under NaCl and KCl stress of were partially restored.This not only indicates that GmCLCs genes of soybean respond significantly to NaCl stress,but also implies that has an important effect on the regulation of Cl-transport in yeast.On the basis of previous functional verification and screening,the apparent alleviation function and its molecular mechanism of GmCLC1 and GmCLC-c2 have been studied intensively.When the GmCLC1 was overexpressed in soybean hairy root composite plants and Arabidopsis,it reduced the accumulation of Cl" in shoots and alleviated the adverse effects on growth of Arabidopsis seedlings and soybean hairy root composite plants under salt stress.Through overexpression GmCLC-c2 in Arabidopsis thaliana and the mutant atclcb,we found that GmCLC-c2 directly regulates the distribution and content of Cl-and NO3-in seedlings;for example,it reduced the ratio of Cl-/NO3-in arabidopsis thaliana seedlings,and indirectly affected the Na+and K+ contents,so that the seedlings showed a mitigation effect on salt stress.In addition,we used Xenopus oocytes to establish a heterologous expression system and expression of soybean GmCLC-b1 and GmCLC-c2 proteins in Xenopus oocytes.The ion channel activity of soybean GmCLC-bl and GmCLC-c2 coding proteins were examined by electrophysiological experiments.Through comparing the current changes in Xenopus oocytes before and after the addition of anion channel inhibitor5-nitro-2-(3-amphetamine)benzoic acid(NPPB),as the voltage increases the current increases exponentially compared with the negative control before adding NPPB.However,when NPPB was added,the current decreased significantly and was almost no difference with the negative control.It indicates the chloride channel properties of GmCLC-bl and GmCLC-c2 encoding proteins.In a word,we screened G/mCLC1 and GmCLC-c2 genes of soybean that has strong tolerance to(chlorine)salt stress by technology of soybean hairy root composite plants,the quantitative real-time PCR and yeast complementation.The molecular mechanism that GmCLCl and GmCLC-c2 genes of soybean whose could significantly alleviate the salt stress by reducing the Cl-content in seedling shoots was further explained via the technology of overexpression in Arabidopsis thaliana and electrophysiological. |
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