Comparative Study On Salt And Drought Resistance Between Transgenic Alfalfa Lines Overexpressing ZxNHX1 From Zygophyllum Xanthoxylum And AtNHX1 From Arabidopsis

Zygophyllum xanthoxylum is a perennial woody xerophytic shrub with abundant resistance gene resources.Previous studies found that under salt or drought stress,the expression of Na~+/H~+antiporter gene ZxNHX1 was positively correlated with the accumulation of Na~+.Furthermore,overexpressed ZxNHX and ZXVP1-1 genes were polymerized into alfalfa,the drought and salt tolerance of the transgenic lines were significantly improved,and transgenic lines accumulated more cations,especially Na~+.However,recent studies have found that AtNHX1 of Arabidopsis thaliana may mainly mediate the sequestration of K~+,indicating that the function of the vacuolar membrane Na~+/H~+antiporter may be different among different plants.Our previous study using yeast as well as amino acid sequences alignment analysis showed that there may be differences in the selectivity of Na~+and K~+between ZxNHX1 from xerophyte Z.xanthoxylum and AtNHX1 from model plant A.thaliana,but it has not been verified at the whole plant level.In this study,therefore,we transferred the ZxNHX1 and AtNHX1 into alfalfa respectively,and obtained the transgenic lines with similar expression levels of ZxNHX1 and AtNHX1 by molecular identification.Then the differance on functions of ZxNHX1 and AtNHX1 was analyzed at whole plant level by comparing the resistance and ion accumulation pattern between different transgenic alfalfa lines.The main results are as follows:1.A total of 9 resistant ZxNHX1-transferred plants,4 resistant AtNHX1-transferred plants and10 resistant empty vector-transferred plants were obtained by Agrobacterium infecting the leaves of alfalfa cultivar Xinjiang Daye,respectively.These resistant plants were identified by PCR,and it was found that the positive rate was 100%,indicating that foreign genes have been integrated into the the genome of alfalfa.Then,the expression of foreign genes in 9 ZxNHX1 transgenic lines and4 AtNHX1 transgenic lines were identified by semi quantitative RT-PCR and qRT-PCR,two transgenic lines with relatively consistent gene expression levels were selected(referred to as:A1,A2,Z1,Z2),which thus were used for subsequent analysis.2.The salt tolerance of alfalfa were improved by overexpression of either AtNHX1 or ZxNHX1.Compared to AtNHX1,however,the overexpression of ZxNHX1 confers stronger salt tolerance to transgenic alfalfa.After 200 mmol/L NaCl treatment for 15 d,the growth of wild type and empty vector control line were severely inhibited with leaves wilting,while all of NHX transgenic alfalfa lines still showed normal growth.By comparison,the growth status of the transgenic lines overexpressing ZxNHX1 was better than that of the transgenic lines overexpressing AtNHX1.Under200 mmol/L NaCl,the plant height,shoot fresh weight,,relative leaf water content and the net photosynthetic rate of Z1 were 10.9%,11.9%,16.0%,7.6%and 20.5%higher than that of A1,respectively.3.Overexpression of ZxNHX1 conferred stronger drought in transgenic alfalfa compared to that of AtNHX1.After being treated with drought stress(field water capacity:30%)for 20 days,both of the wild type and empty vector control plants showed wilting,their growth was severely suppressed,and even died,but four transgenic lines maintained steable growth.Moreover,the growth of Z1 was better than A1 with higher plant height,shoot fresh weight,and relative leaf water content by 13.3%,30%,and 22.5%,respectively,under drought condition..4.There were different Na~+and K~+accumulation characteristics between transgenic alfalfa lines overexpressing AtNHX1 or ZxNHX1.After treated with 200 mmol/L NaCl for 15 days,the Na~+concentration in leaves,stems and roots of Z1 was 27.3%,17.9%,and 29.1%higher than that of A1,while the K~+concentration in leaves,stems and roots of latter was significantly higher compared to that of former by 26.2%,17.5%and 27.6%,respectively.Similarly,after drought stress for 20 days,the Na~+concentration in Z1 plants showed significantly higher than that of A1 plants,while K~+accumulation in the latter was higher than that in the former.These results suggested that the overexpression of Z.xanthoxylum ZxNHX1 was more effective to enhance the Na~+accumulation ability of the transgenic alfalfa,while the overexpression of A.thaliana AtNHX1 preferentially promoted the K~+accumulation of transgenic plants.