Identification Of CLC In Camellia Sinensis And Functional Verification Of CsCLCe

Fluoride usually has adverse effects on plant morphology,growth and development.Tea plant(Camellia sinensis(L.)O.Kuntze)as a hyper-accumulator of F compared to other plants,but its tolerance mechanism remains unclear.It has been recently found that the Voltage-gated Chloride Channel(CLC)superfamily can transport Cl-,NO3-,F-and other anions.Some studies subsequently have shown that bacterial CLCs have the function of exporting F-from cytoplasm to prevent fluoride toxicity.In order to explore the mechanism of fluoride accumulation in C.sinensis,in this article we identified CsCLCs genes and analyzed them by bioinformatics.We also analyzed expression levels of CsCLCs genes in different tissues after different concentrations of Cl-and F-treatment.In order to provide evidence of F accumination mechanism and a theoretical basis for the genetic improvement of tea plants,we selected a CsCLCe gene that may have function of fluoride transport and verified the function of CsCLCe under fluoride stress.The main results are as follows:1.Eight CsCLCs genes were identified from the CSS genome(accession no.GS200008),and named CsCLCb-e,CsCLCfl-f2,CsCLCgl-g2,respectively.The number of exons in CsCLCs were 3 to 24,and all CsCLCs proteins contained 2 to 10 transmembrane domains.A phylogenetic tree was classified CsCLCs proteins into two subclasses,the CsCLCs members in subclass II(CsCLCb-d、CsCLCg1-g2)were contained three conserved residues GxGIPE(Ⅰ),GKxGPxxH(Ⅱ)and PxxGxLF(Ⅲ),while members in subclass I showed the different conserved residues.These results provide some basic informations for further understanding and analysing fluoride accumulation genes in CsCLCs members.2.When tea plants were treated by different concentrations of chloride(0,5,10 mM)and fluoride(0,0.14.0.28 mM),the expression levels in different tissues results showed that the response of CsCLCs to F-and Cl-was sub-functionalized.It was found that CsCLCc and CsCLCg2 in subclass II were more sensitive to Cl-treatment and may participate in absorption and long-distance transport of Cl-in tea plants.CsCLCs members in subclass I(CsCLCe、CsCLCfl-f2)were significantly responsive to fluoride treatment compared to members in subclass II(CsCLCb-d、CsCLCgl-g2).3.CsCLCe gene was cloned from C.sinensis and may be related to fluoride transport.The open reading frame(ORF)of CsCLCe is 2355 bp,which encodes 784 amino acids.CsCLCe was closely related to the evolution of bacteria CLCFs,and conserved sequences in CsCLCe were different from conserved sequences in subclass Ⅱ.In addition,the subcellular localization results of overexpressed CsCLCe-type Arabidopsis roots verified that CsCLCe was localized on plasma membrane.The role of CsCLCe in fluoride transport was verified by analyzing overexpressed CsCLCe-type Arabidopsis under fluoride stress.It was verified that the germination rate and root length in overexpressed lines were significantly better than those of wild type.It was proved that the overexpressed lines had stronger resistance to fluoride comparing with wild-type Arabidopsis.In summary,bioinformatics methods were used to study the basic information about CsCLCs superfamily in this article.Combined with the relative expression of CsCLCs under the treatment of fluoride and chloride,the CsCLCe gene that may have the function of fluoride transport was selected to verify its fluoride tolerance.The experimental results not only provide basic information for understanding the members of CsCLCs,but also provide data for exploring the molecular mechanism of fluoride accumulation in C.sinensis.