Molecular Identification Of ZxNHX1-VP1-1 Co-overexpressing Alfalfa,and The Evaluation Of Salt Tolerance In Alfalfa Co-overexpressing ZxNHX1-PcCLCg

Alfalfa(Medicago sativa L.)is one of the most important legume forages with high protein,low fiber,and good palatability,which is known as the“king of forage”.However,most alfalfa cultivars are difficult to maintain high yields in arid and semi-arid areas and saline environment in northwest China.Therefore,it was expected to improve the stress resistance of alfalfa by transferring excellent stress resistance genes of desert plants into alfalfa.Zygophyllum xanthoxylum have both strong drought and salt tolerance,its tonoplast Na~+/H~+antiporter encoding gene ZxNHX1 and tonoplast H~+-pyrophosphatase encoding gene ZxVP1-1 are both key genes for vacuolar Na~+compartmentalization for both salt and drought resistances.We have previously cloned ZxNHX1,ZxVP1-1 with herbicide resistance gene Bar,and transferring into alfalfa.The co-overexpressing ZxNHX1-VP1-1 alfalfa were obtained,but its molecular characteristics have not been clear.In addition,in saline environment,in addition to Na~+,excessive accumulation of Cl~-in plant cytoplasm can also produce toxic effects.However,the current researches mainly focus on Na~+,and there are few studies on Cl~-.The desert plant Pugionium cornutum is a typical plant which accumulates chlorine.Our previous studies showed that the vacuolar membrane Cl~-channel gene PcCLCg is a key gene for the resistance to chlorine in Pugionium cornutum,and polymerized PcCLCg with ZxNHX1 co-transferring into alfalfa and obtained the resistant plants.But its salt tolerance has not been evaluated.In this study,PCR,RT-PCR,Southern Blot,Western Blot and ELISA were used for exploring the molecular characteristics of the co-overexpressing ZxNHX1-VP1-1alfalfa.And the salt tolerance of co-overexpressing ZxNHX1-PcCLCg alfalfa and overexpressing ZxNHX1 alfalfa were evaluated and compared.The main results of this study are as followings:1.By PCR identification,a total of 8 alfalfa positive plants co-overexpressing ZxNHX1-VP1-1 genes were obtained.The target genes ZxNHX1,ZxVP1-1 and the marker gene Bar of No.8 transgenic alfalfa were inserted into single copy number by Southern Blot identification.Western Blot and ELISA showed that ZxNHX1 and ZxVP1-1 proteins could be expressed normally in No.8 transgenic alfalfa,and the expression of these two proteins were the highest in leaves,followed by stems and roots.These results indicated that the exogenous ZxNHX1、ZxVP1-1 genes had been successfully transferred into the alfalfa genome and could be expressed normally at the protein level.2.By PCR identification,a total of 16 alfalfa positive plants co-overexpressing ZxNHX1-PcCLCg genes were obtained.Co-overexpressing ZxNHX1-PcCLCg alfalfa and overexpressing ZxNHX1 alfalfa were analyzed by RT-PCR.One co-overexpressing strain(NC-1 and NC-2)with high expression of ZxNHX1 and PcCLCg genes and one low expression of ZxNHX1 and PcCLCg genes were screened,and one overexpressing strain(N-1)with the same expression of ZxNHX1 as that in NC-1 was used for subsequent salt tolerance analysis.3.After 50 and 200 mmol/L NaCl treatments for 10 days,the plant height,shoot fresh and dry weight of NC-1 line was significantly higher than N-1 line,empty vector and wild type.These results showed that the co-overexpression of ZxNHX1 and PcCLCg genes could enhanced the salt tolerance of alfalfa.4.Under salt treatment,the contents of Na~+and Cl~-in each strain increased gradually,while the contents of K~+decreased gradually.Under 50 mmol/L NaCl treatment,the Na~+content in leaves and roots of NC-1 line was significantly higher than NC-2 and N-1 lines,respectively;the content of Cl~-in leaves and stems of NC-1line was significantly higher than N-1 line,empty vector and wild type.Under 200mmol/L NaCl treatment,the Na~+content in leaves and stems of NC-1 line was significantly higher than NC-2,N-1 lines,empty vector and wild type,and the Na~+content in roots of NC-1 and N-1 lines was significantly higher than NC-2 line,empty vector and wild type.The content of Cl~-in leaves,stems and roots of NC-1 line was significantly higher than NC-2 and N-1 lines,respectively.These results showed that the co-overexpression of ZxNHX1 and PcCLCg genes enhanced the accumulation of Na~+and Cl~-in the leaves of alfalfa.5.Under the control condition(0 mmol/L NaCl),the contribution of Na~+to leaf osmotic potential of all lines was low and there was no significant difference.Under50 and 200 mmol/L NaCl treatments,the contribution of Na~+and Cl~-to leaf osmotic potential in NC-1 line was significantly higher than NC-2 and N-1 lines.Consistent with Na~+,Cl~-contributed less to leaf osmotic potential in all lines under the control conditions.Under 50 mmol/L NaCl treatment,the contribution of Cl~-to leaf osmotic potential in NC-1 line was significantly higher than NC-2 and N-1 lines,respectively.Under 200 mmol/L NaCl treatment,the contribution of Cl~-to leaf osmotic potential in NC-1 and NC-2 lines was significantly higher than wild type and empty vector.These results showed that co-overexpression ZxNHX1 and PcCLCg alfalfa could increase the osmotic regulation ability of transgenic plants under salt stress by accumulating Na~+and Cl~-.6.Under salt treatment,the relative plasma membrane permeability of N-1 line,empty vector and wild type increased gradually with the increase of salt concentration,while the relative plasma membrane permeability of NC-1 and NC-2 lines remained stable.Under 200 mmol/L NaCl treatment,the leaf osmotic potential of NC-1 was significantly lower than NC-2,N-1 lines,empty vector and wild type.