Characterization Of Cation/Proton Antiporter Family And Functional Validation Of RsNHX1 And RsNHX2 Genes Involved In Salt Tolerance Of Radish(Raphanus Sativus L.)

Radish(Raphanus sativus L.,2n=2x=18),originating from China,is one of the root vegetable crops with high nutritional and medicinal value.As an important economic crop,the radish has been widely cultivated wordwide.Salt stress is one of the abiotic stresses in crop production,which has a serious impact on crop quality and yield.Fleshy taproot of radish is the main edible organ,and different radish genotypes have different sensitivity to salt stress.Therefore,it is imperative to improve the salt tolerance of radish for genetic breeding.In recent years,the comprehensive isolation and identification of a range of gene families has become popular with the rapid development of plant genome sequencing.CPA(Cation proton antiporters)family is involved in the transport and exchange of monovalent cation,which plays important roles in stress tolerance as well as ionic and pH homestasis.Although the identification of CPA family has completed in several plant species,the molecular characterization of CPA family still remains unclear in radish.In addition,NHX(Na+/H+Exchanger)gene family,the subfamily of CPA,it is closely related to salt stress response and has been well studied in Arabidopsis thaliana,but the research in radish is still lacking.In this study,the candidate CPA genes were identified from the radish genome sequences and the expression pattern of CPA genes were verified by RNA-seq and real time-quantitative PCR approach.The overexpression of RsNHX1(RsCPA31)and RsNHX2(RsCPA32)genes were conducted to validate the biological functions in Arabidopsis thaliana.The main results were as follows:1.Based on the radish genome sequnces,a total of sixty CPA candidate genes containing Na+/H+exchanger domain were identified.Phylogenetic analysis indicated that the RsCPA genes could be divided into three classes including 9 NHX(Na+/H+ Exchanger)genes,10 KEA(K+Efflux Antiporter)genes and 41 CHX(Cation/H+Exchanger)genes.Among them,58 RsCPA genes(96.67%)were located in radish chromosome R1-R9.Gene structure analysis showed that RsNHX and RsKEA genes were more complex than RsCHX genes.Protein prediction showed that the molecular weight of RsCPA protein was ranged from 25 to 126 kDa.The instability coefficient was 37.02(<40.00),indicating that most of RsCPA protein structures were stable.The aliphatic index indicated that the RsCPA proteins contained a large number of fatty acids,and all of the RsCPA proteins are predicted to be hydrophobic proteins.Motifs analysis of RsCPA proteins showed that motif 5,motif 6 and motif 11 were widespread in CHX,KEA and NHX families,confirming these motifs were conserved in RsCPA proteins.2.Based on the RNA-Seq transcriptome data reported in previous study,some RsCPA genes showed spatio-temporal expression profiles in different tissues and stages.RT-qPCR analysis indicated all of the NHX genes were significantly up-regulated with 250 mM-L-1 NaCl treatment.The expression level of RsCPA13,RsCPA29,RsCPA31 and RsCPA35 was peak in 24h with NaCl treatment,and the expression level of RsCPA21 and RsCPA34 was peak in 12h.However,The expression level of RsNHXs were subsequently maintained at the normal level after 96h with NaCl treatment,which indicated that NaCl could induce the expression of NHX genes.3.RsNHX1(RsCPA31)and RsNHX2(RsCPA32)genes were cloned from the ’NAU-XBC’ variety,and then the over expression vector pCAMBIA-RsNHX1 and pCAMBIA-RsNHX2 were constructed.The Arabidopsis plants were infected by agrobacterium-mediated floral dip method.The positive plants were screened by 1/2 MS culture medium containing 25 mg-L’1 Kan.RsNHX1 and RsNHX2 genes has been successfully verified by PCR.Wild-type Arabidopsis thaliana and transgenic plants were treated with 200 mM-L-1 NaCl,and the transgenic lines were significantly resistant to salt than WT.Phenotypic identification preliminarily verified that RsNHX1 and RsNHX2 was involved in salt tolerance of plants.These results could lay theoretical foundation for clarify the biological roles of RsNHX1 and RsNHX2 genes involved in the regulatory networks of salt stress response in plants.