Cloning Of The MsNCL1 Gene In Alfalfaand Its Function In Response To Saline-alkaline Stress(Medicago Sativa L.)

Saline-alkali stress is one of the major abiotic stresses that restrict crop production worldwide.Alkaline salt is mainly used in saline-alkali land of Songnen Plain,and its main components are Na HCO₃and Na₂CO_3.Na⁺is the most abundant soluble salt in saline-alkali soil.Excessive accumulation of Na⁺is harmful to most crops,so maintaining the steady state of Na⁺is the key to salt tolerance of crops.NCL transporters usually utilize calcium ion gradients in plant cells to reduce the absorption of sodium ions through internal flow of calcium ions,thereby improving the salt and alkaline tolerance of plants.In this study,the Ms NCL1 gene of Medicago sativa was cloned and transformed into Arabidopsis thaliana,and the function of Ms NCL1 gene of Medicago sativa was verified by analyzing the phenotypic,ionic components and physiological changes of the transformed Arabidopsis thaliana under saline-alkaline conditions.The main research results are as follows:1.Cloning and bioinformatics analysis of Ms NCL1 geneThe Ms NCL1 gene of alfalfa was cloned by RT-PCR.The gene has a total length of2133 bp and encodes 710 amino acids.It contains three EFh domains between amino acids 350 to 378,448 to 476 and 485 to 513 of the protein sequence,and one Na-Ca-ex domain between amino acids 556 to 702.Ms NCL1 protein had 11 transmembrane domains,belonging to the stability protein.Its secondary structure was as follows:α-helix accounted for 53.94%,extended chain accounted for 13.66%,β-turn accounted for 5.21%,and irregular curl accounted for 27.18%.Evolutionary tree analysis showed that Medicago sativa Ms NCL1 was the closest relative to Medicago truncatula Mt NCL1,and sequence alignment indicated that there existed differential sites between Ms NCL1and Mt NCL proteins,four amino acid differential sites in the EF-hand structure,and two amino acid differential sites in the Na-Ca-ex domain.2.Abiotic stress expression and tissue-specific expression of Ms NCL1 geneUnder saline-alkali treatment,Ms NCL1 expression showed an upward trend,which was higher than that in the control group,and the expression was the highest at 3 h.Under low temperature of 4℃,Ms NCL1 expression showed a downward trend at the other time points,except that there was no significant change for 7 d.Under salt treatment,Ms NCL1 gene expression was up-regulated all except for 6 h,and the highest expression was observed at 7 d,which was about 23 times that of the control group.Under drought treatment,Ms NCL1 gene showed an upward trend at 6 h and 12 h,and there was no significant change at other time points.Ms NCL1 gene was expressed in root,stem,leaf,flower and pod of Medicago sativa,and the highest expression was in the pod and the lowest in the root.3.Construction of PEG（Pearly Gate）-Ms NCL1-1Xflag Expression Vector and Genetic Transformation of Arabidopsis thalianaFirst,the Ms NCL1 gene obtained by cloning was connected with an intermediate carrier p GWC to obtain a plant intermediate carrier p GWC-Ms NCL1,which was then connected with an expression carrier p EG（pearly gate）to construct a p EG（pearly gate）-Ms NCL1-1XFlag plant expression carrier,The Ms NCL1 gene was introduced into Arabidopsis thaliana,and 10 transgenic Arabidopsis thaliana plants were screened and obtained combined with PPT resistance screening and PCR detection.The results of q RT-PCR assay showed that the expression levels of the target gene were the highest in Lines 6 and 9.Three primers were used for homozygous identification of arabidopsis mutant ncl147.The results showed that ncl147 was a homozygous mutant.4.Resistance analysis of arabidopsis thaliana transform with Ms NCL1 gene（1）phenotypic analysis of arabidopsis thaliana transform with Ms NCL1 gene under saline-alkaline stressThe whole plant phenotype and plate root length of arabidopsis thaliana transformed with Ms NCL1 gene under 150 m M Na HCO₃treatment were studied.The results showed that the root length of the upright plate of arabidopsis thaliana transformed with Ms NCL1 gene was significantly higher than that of WT and ncl147mutants,and it showed strong salt and alkaline tolerance.The whole plant phenotype of arabidopsis thaliana transform with Ms NCL1 gene was tested.The results showed that the leaves of Arabidopsis thaliana transformed with Ms NCL1 gene only slightly wilted,while the leaves of WT and ncl147 mutants showed more severe wilting.This indicated that the overexpression of Ms NCL1 gene enhanced the resistance of transgenic arabidopsis to saline-alkali.（2）Analysis of physiological index of arabidopsis thaliana transformed with Ms NCL1 gene unde saline-alkaline stressUnder saline-alkali stress,the MDA,O₂-and H₂O₂contents of Arabidopsis N6 and N9 transgenic with Ms NCL1 gene were lower than those of WT and ncl147 mutants,and the proline,soluble sugar and soluble protein contents were higher than those of WT and ncl147 mutants.At the same time,the DAB and NBT staining experiments under saline-alkaline stress showed that the coloration levels of Arabidopsis N6 and N9transfected with Ms NCL1 gene were significantly lower than those of WT and ncl147mutants,indicating that Arabidopsis N6 and N9 transfected with Ms NCL1 gene accumulated less active oxygen substances compared with WT and ncl147 mutants.The results above indicated that Ms NCL1 gene could enhance the plant resistance to saline-alkali stress.（3）Ion analysis of arabidopsis thaliana transform with Ms NCL1 gene under saline-alkali stressUnder saline-alkali stress,the Ca²⁺ion contents of Arabidopsis N6 and N9transgenic with Ms NCL1 gene were higher than those of WT and ncl147 mutants,while Na⁺ion content and Na⁺/K⁺were lower than those of WT and ncl147 mutants.At the same time,NMT experiments under saline-alkaline stress showed that the Ca²⁺influx of Arabidopsis thaliana transgenic with Ms NCL1 gene was significantly higher than that of WT and ncl147 mutants,and the Na⁺absorption degree was significantly lower than that of WT and ncl147 mutants,indicating that Arabidopsis thaliana transgenic with Ms NCL1 gene had better Ca²⁺influx and less Na⁺absorption flow rate than WT and ncl147 mutants.The results above indicated that Ms NCL1 gene might enhance plant resistance to saline-alkali stress by regulating calcium and sodium ion transport.