Ion Selectivity Analysis Of ZxCNGC1;2 And ZxCNGC2;1-ZxCNGC4 Complex From Zygophyllum Xanthoxylum

Zygophyllum xanthoxylum is a succulent xerophyte that grows in the desert areas of northwest China,which has formed a unique stress resistance mechanism in the long-term evolutionary process.Studies have shown that Z.xanthoxylum can absorb trace amounts of Na⁺in the soil as a beneficial element,and accumulate a large amount in vacuole to resist osmotic stress.It can be seen that in-depth study of the Na⁺transport mechanism in Z.xanthoxylum is of great significance for elucidating its drought resistance.Cyclic nucleotide-gated ion channels（CNGCs）play an important regulatory role in plant growth and development,biotic and abiotic stress responses.We cloned three ZxCNGC genes in Z.xanthoxylum,named ZxCNGC1;1,ZxCNGC2;1and ZxCNGC4.The results show that ZxCNGC1;1 can mediate the transport of Na⁺,Ca²⁺and Mg²⁺.However,the transport activities of Na⁺could not be detected in ZxCNGC2;1 or ZxCNGC4.In this study,the ZxCNGC gene family was bioinformatically analyzed using the Z.xanthoxylum genome data,and the expression pattern of ZxCNGC1s was analyzed by RT-qPCR.ZxCNGC1;2 was obtained by screening,and its function was cloned and explored.In addition,the interaction of ZxCNGC2;1 and ZxCNGC4 were identified and their ion transport activities were analyzed.The following main results were achieved:1.A total of 24 ZxCNGCs genes were identified in the Z.xanthoxylum genome,all of which contained typical CNBD conserved domain.They were divided into 5subfamilies,randomly distributed on 11 chromosomes of Z.xanthoxylum,with the largest distribution on chromosome 6.Gene structure analysis showed that ZxCNGCs have 5 to 11 introns and 6 to 12 exons.There are 9 members of the ZxCNGC1 gene family,among which ZxCNGC1;1 and ZxCNGC1;2 have high homology with At CNGC1.ZxCNGC1;1 and ZxCNGC1;2 are mainly expressed in the roots,and the expression abundance of ZxCNGC1;2 was up-regulated about 2-fold in roots after treated with 50 m M NaCl for 6 h.2.ZxCNGC1;2 has a coding sequence of 2136 bp long and belonged to the CNGCs I subfamily of higher plants.The encoded protein was subcellularly localized to the cytoplasmic membrane.The expression abundance of ZxCNGC1;2 under salt treatment showed a trend of increasing,then decreasing and then increasing,reaching a peak at 150 m M NaCl treatment for 6 h.GUS staining showed ZxCNGC1;2 was mainly expressed in the mature region of Z.xanthoxylum root.In yeast heterologous expression system,ZxCNGC1;2 has Na⁺transport activity,but could not detect K⁺transport activity;OsCNGC1 could not detect Na⁺or K⁺transport activity.In HEK293T cell heterologous expression system,both ZxCNGC1;2 and OsCNGC1 can mediate Na⁺,Ca²⁺and Mg²⁺transport,but K⁺transport activity could not be detected.In addition,the transport capacity of ZxCNGC1;2 for Na⁺was significantly stronger than that of OsCNGC1,and the transport capacity of OsCNGC1 for Mg²⁺was significantly stronger than that of ZxCNGC1;2.3.The results of bimolecular fluorescence complementation and yeast two-hybrid experiments indicated that ZxCNGC2;1 and ZxCNGC4 can interact with each other.However,no Na⁺or K⁺transport activity of ZxCNGC2;1-ZxCNGC4 complex was detected in the yeast heterologous expression system.In the HEK293T cell heterologous expression system,both ZxCNGC2;1 and ZxCNGC4 had Ca²⁺and Mg²⁺transport activities,but neither Na⁺or K⁺transport activity was detected.ZxCNGC2;1-ZxCNGC4 complex had Na⁺,Ca²⁺and Mg²⁺transport activities,but no K⁺transport activity was detected.It shows that ZxCNGC2;1-ZxCNGC4 complex can form a new ion channel through interaction,which is able to transport Ca²⁺and Mg²⁺with an increased Na⁺transport activity.The above results confirm ZxCNGC1;2,ZxCNGC2;1-ZxCNGC4 complex can participate in Na⁺transport,revealing the unique Na⁺transport pathway,which lays a foundation for further elucidating the resistance mechanism of Z.xanthoxylum.