Cloning And Functional Analysis On ZxCNGC1?ZxCNGC2 And ZxCNGC4 From Zygophyllum Xanthoxylum

Drought and soil salinization have become two abiotic stress factors that seriously hinder the sustainable development of agriculture and animal husbandry.However,plants growing in the desert region of Northwest China have evolved unique resistance mechanisms in response to the extremely harsh living environment.Therefore,it is beneficial to the genetic improvement of crops and fine forage grass to explore the stress-resistant gene resources of desert plants in Northwest China.Zygophyllum xanthoxylum is a kind of succulent xerophyte,which is the dominant species of desert vegetation in Northwest China,and has strong salt tolerance and drought resistance.Previous laboratory researches found that the Na⁺content in soil of Z.xanthoxylum in Alxa desert was 1/66 of that in soil salinization of Suaeda salsa in Hexi Corridor,but the Na⁺content in leaves of Z.xanthoxylum was similar to that in S.salsa,indicated that Z.xanthoxylum could uptake a large amount of Na⁺from low-salinity soil to adapt to arid region.In addition,50 m M Na Cl could significantly improve the drought tolerance and stimulate the growth of Z.xanthoxylum under osmotic stress.Therefore,Na⁺plays a vital role in the process of Z.xanthoxylum resistance to adversity.The molecular mechanism of Na⁺uptake in Z.xanthoxylum was previously studied by our research group.The results showed that Zx HKT1;2 could mediate the uptake of Na⁺in roots.Zx SOS1 could transport a large amount of Na⁺absorbed from the roots to the shoot.Loading Na⁺into mesophyll cells from the ectoplasmic space mediated by Zx HKT1;3.Compartmenting the excess Na⁺in mesophyll cells into vacuoles mediated by Zx NHX1,which could be used as a beneficial osmotic regulator.Zx HKT1;1 could unload Na⁺from xylem sap into xylem parenchyma cells,to reduce the content of Na⁺in leaves,and finally reach the state of ion homeostasis.Electrophysiological analysis showed that Zx HKT1;2 acts as a high affinity transporter.However,Na⁺uptake in plant roots is usually mediated by low affinity channel proteins under high salinity conditions.Plant CNGCs,as non-selective cationic channel proteins,could pass through K⁺,Na⁺and Ca²⁺,and CNGCs are considered to be one of the ion channels involved in Na⁺influx under high salt conditions.At present,the role of CNGCs channel proteins in the Na⁺absorption and ion homeostasis of Z.xanthoxylum is still unclear.In view of this,the cyclic nucleotide-gated channel protein genes encoding Zx CNGC1,Zx CNGC2 and Zx CNGC4 were cloned from Z.xanthoxylum.Revealing the expression patterns in Z.xanthoxylum by the q RT-PCR.Analyzing the subcellular localize by the transient expression of fluorescence fusion protein in tobacco.Analyzing the ion-selective characteristics by using heterologous expression systems.The cell-specific expression was performed by in situ PCR and GUS staining.The function of Zx CNGC1,Zx CNGC2 and Zx CNGC4 in the process of Z.xanthoxylum resistance was preliminarily investigated,and the overall results can be summarized as follows:1.The open reading frame of Zx CNGC1 is 2133 bp,and has six transmembrane domains.It belongs to the subfamily I of higher plant CNGCs and the subcellular located in the plasma membrane.2.In yeast and HEK293T cells,Zx CNGC1 not only mediates the influx of divalent cation Ca²⁺and Mg²⁺,but also mediates the influx of monovalent cation Na⁺.At CNGC1can only mediate Ca²⁺and Mg²⁺influx.3.Zx CNGC1 was mainly expressed in epidermis and cortical cells in the root maturation region,and the expression was up-regulated by 100 m M Na Cl.4.The open reading frames of Zx CNGC2 and Zx CNGC4 were 2094 and 2040 bp,respectively,with 6 transmembrane domains.Both belong to group b of subfamily?of higher plant CNGCs,and the subcellular localized in the plasma membrane.5.The analysis of the yeast heterologous expression system showed that the absorbing ability for Na⁺and K⁺of Zx CNGC2 and Zx CNGC4 has not been analyzed.6.Both Zx CNGC2 and Zx CNGC4 were mainly expressed in the leaves of Z.xanthoxylum,and their expression levels were induced by 50 and 100 m M Na Cl.These results indicated that Zx CNGC1 was mainly localized in epidermis and cortex of roots,and the expression was up-regulated by salt stress.It may act as a low affinity channel protein to mediate the uptake of Na⁺in roots.At the same time,it also mediates the absorption of Ca²⁺and Mg²⁺.The expression levels of Zx CNGC2 and Zx CNGC4 were up-regulated by salt treatment,suggesting that they may involve in salt stress response.However,the absorption capacity of Zx CNGC2 and Zx CNGC4 on Na⁺and K⁺was not analyzed at the cellular level.