Cloning And Salt Tolerance Analysis Of An Aquaporin Gene(MsPIP2;2) From Alfalfa(Medicago Sativa.L)

Alfalfa(Medicago sativa.L)is a perennial high-quality leguminous forage that is widely cultivated throughout the world.It can improve stress resistance through symbiosis with rhizobium in the soil.Alfalfa is praised as the 'king of forage' because of its wide range of cultivation,high nutritional value and good palatability.Although alfalfa has strong resistance to obiotic stress,adverse environmental factors such as drought,salt,low temperature,high temperature,diseases and pests can still cause a serious decline in the yield and quality of alfalfa.With the development of molecular biology,genetic improvement of alfalfa by transgenic technology has been widely used,which also provides a theoretical basis for breeding new alfalfa varieties with high quality and high resistance.Aquaporins(AQPs)are membrane channel proteins that responsible for water transport in plants.It is very important to maintain water balance in plants and can participate in various physiological processes in plants.However,most AQPs functions are still poorly understood in the plant kingdomIn this study,the full-length cDNA sequence of MsPIP2;2 from alfalfa was cloned and isolated by RACE technique.MsPIP2;2 sequence was analyzed by bioinformatics software The tissue expression pattern of MsPIP2;2 gene was analyzed by qRT-PCR and the protein subcellular localization was performed by injecting tobacco leaves.In addition,MsPIP2;2 transgenic Arabidopsis and alfalfa plants were obtained through the construction of overexpression vector pCAMBIA1300-MsPIP2;2-GFP,knockout vector pCRISPR/Cas9-Csy4-MsPIP2;2 and Agrobacterium-mediated transgenic technology MsPIP2;2 function was analyzed using transgenic plants.The main results are as follows:1.The full-length cDNA sequence of MsPIP2;2 was cloned from alfalfa by RACE technique.The sequence analysis showed that the full-length cDNA of MsPIP2;2 was 1322 bp with an open reading frame of 864 bp nucleotides,encoding a protein of 287 amino acids residues,and the GenBank accession number was MK109796.Amino acid multiple sequence alignments indicated that MsPIP2;2 was highly homologous to PIP2;2 from other legumes and contained the same conserved domain(2 NPA motifs and 6 transmembrane helixes).An unrooted phylogenetic tree was constructed using neighbor-joining method revealed that MsPIP2;2 was clustered into a branch with other legume plants and shared the highest homology with Medicago truncatula,illustrating the evolutionary conservation of MsPIP2;2 protein2.The tissue expression pattern of MsPIP2;2 gene was examined by qRT-PCR.The results showed that MsPIP2;2 was expressed in both roots and leaves,and there was no significant difference in expression abundance between them.To confirm the response of MsPIP2;2 to abiotic stress,the transcript levels were determined after different treatments.MsPIP2;2 expression was not significantly altered under drought treatment,while the transcript levels were significantly upregulated in roots and leaves under salt stress and ABA treatment.3.To test the subcellular localization of MsPIP2;2,the MsPIP2;2-GFP fusion protein(pCAMBIA1300-MsPIP2;2-GFP)and pm-rk(a plasma membrane marker)were transiently co-expressed in tobacco leaves via Agrobacterium-mediated transformation.Our results showed that the MsPIP2;2 protein was localized mainly in the plasma membranes of plant cells.4.MsPIP2;2 gene was overexpressed in Arabidopsis by the floral dip method and homozygous lines(T3 generation)were generated successfully.Functional studies indicated that MsPIP2;2 could regulate physiological and biochemical processes of transgenic plants by reducing the membrane damage and ROS accumulation,increasing the activity of antioxidant enzymes SOD,POD and CAT,proline content and maximum photosynthetic efficiency Fv/Fm.MsPIP2;2 also alleviated salt toxicity by promoting Na+efflux and K+retention in transgenic plants.In addition,MsPIP2;2 improved plant salt tolerance by regulating the expression of endogenous stress-responsive genes.5.By adjusting the Ca2+concentration and pH value in the medium,it was found that high concentration Ca2+(?6mM)or low pH(?5)enhanced the salt tolerance of MsPIP2;2 transgenic Arabidopsis thaliana.However,there was no significant difference between transgenic plants and WT under salt stress with low Ca2+(?1.5 mM)or high pH(?6.5)conditions.6.MsPIP2;2 gene knockout vector was successfully constructed using CRISPR/Cas9 technology.MsPIP2;2 overexpression and knockout transgenic alfalfa was harvested by tissue culture.Overexpression of MsPIP2;2 enhanced salt tolerance of alfalfa by reducing cell membrane damage and oxidative damage caused by ROS accumulation and increasing the accumulation of proline and chlorophyll.In summary,MsPIP2;2 gene was isolated and cloned successfully from alfalfa.MsPIP2;2 expression was mainly induced by salt stress and ABA treatment.MsPIP2;2 protein was localized mainly in the plasma membranes of plant cells.MsPIP2;2 confers salt tolerance of transgenic plants by regulating antioxidant defence system-mediated ROS scavenging,K/Na ion homeostasis and stress-responsive gene expression.