Molecular Mechanism Of Sorghum Overexpressed GsNAC2 Gene Responding To Saline-alkali Stress

Sorghum is the fifth largest food crop in the world and occupies an important position in China’s agricultural production.Osmotic stress,oxidative stress,ion toxicity and high p H caused by saline-alkali stress can seriously affect the growth,and biomass of plants.Therefore,it is of great strategic significance to study the mechanism of plant saline-alkali stress resistance,dig out new genes responding to saline-alkali stress,and cultivate new varieties of saline-alkali tolerance for improving the utilization of saline-alkali soil,and promoting the sustainable development of crops.Transcription factors play an important regulatory role in plant adaptation to stress environment.NAC transcription factors have many functions such as promoting plant growth,and responding to abiotic stress.Wild soybean has strong environmental adaptability,and is an ideal material for cloning stress-resistant genes.It is of great theoretical value to explore the regulatory mechanism of wild soybean GsNAC2 in plant response to saline-alkali stress.Sorghum lines with overexpression of GsNAC2 gene were selected as the research object.After saline-alkali stress treatment（Na HCO₃:Na₂CO₃=5:1,75 m M,p H 9.63）,the molecular mechanism of sorghum with overexpression of GsNAC2 gene in response to saline-alkali stress was determined by measuring growth,physiological,and biochemical indexes,screening the main metabolic processes based on differential transcriptomics data,and analyzing the expression patterns of key genes in plant hormone signal transduction,and glutathione metabolic pathway.The main research results were as follows:（1）GsNAC2 has typical characteristics of NAC family transcription factors.GsNAC2 has the typical NAM domain of NAC family;it has the closest phylogenetic relationship with Gm NAC2,which has been determined to respond to abiotic stress.The N-terminal of amino acid sequence has five highly conserved subdomains,and the C-terminal of amino acid sequence is highly varied.（2）The expression of GsNAC2 gene in sorghum was induced by saline-alkali stress.Twenty-two sorghum lines overexpressing GsNAC2 gene were obtained by agrobacterium tumefaciens-mediated immature embryos of sorghum.The bud length,and fresh weight of sorghum seeds were used as indexes to screen out excellent GsNAC2-11 line with saline-alkali tolerance at bud stage,and the subsequent experiments were carried out with it as material;saline-alkali stress can significantly induce the expression of GsNAC2 gene in sorghum seedlings.GsNAC2 gene is expressed in roots,stems,and leaves of sorghum seedlings,and the highest expression in leaves.（3）Sorghum with overexpression of GsNAC2 gene has strong saline-alkali tolerance.Under saline-alkali stress,transgenic sorghum buds with GsNAC2 gene grew better.Bud length,fresh bud weight,and moisture content were higher than those of control group.Compared with the control group,the relative permeability of cell membrane,and MDA content of transgenic sorghum buds with GsNAC2 gene were less;NBT,and DAB staining degree is lighter,and the range is smaller;CAT,and SOD activities were significantly enhanced.In the control group,the whole seedling was dry,and wilted;the second leaf was dry,and curled,the yellowing was serious,and the root system was brown,and rotten;however,the leaves of transgenic GsNAC2sorghum extended normally,only the tip edge of the leaves turned yellow,and the root browned slightly.Seedling height,chlorophyll content,stomatal conductance,transpiration rate,root activity,soluble protein,soluble sugar,and proline were significantly higher than those of the control group.The relative permeability of cell membrane,and MDA content of GsNAC2transgenic sorghum seedlings were controlled at a relatively low level;there are fewer blue,and brown spots in NBT,and DAB staining of leaves.The activities of POD,CAT,and SOD increased significantly in the middle,and late stage of stress（24-72 h）.（4）A total of 36505 differentially expressed genes were identified by differential transcriptome analysis of sorghum seedlings under saline-alkali stress.COG analysis showed that more differential genes related to defense mechanism were enriched in different stress treatment time;GO analysis showed that there were many differential gene enrichments in biological processes（response to stimulus,small molecule metabolic process）,cell components（cellular anatomical entity,membrane,cell periphery）,and molecular functions（catalytic activity）;KEGG pathway mainly involves plant hormone signal transduction,starch and sucrose metabolism,MAPK signaling pathway,and glutathione metabolism.（5）GsNAC2 gene regulates sorghum response to saline-alkali stress through many ways.In plant hormone signal transduction pathway,there are 20 genes related to ABA signal pathway.After saline-alkali stress,the receptor gene Sb PYL2,positive regulator Sb SAPK9,and Sb ABCI12were up-regulated,while negative regulator Sb PP2C15 were down-regulated.The expression of receptor genes SbChitin1,and SbChitin2 was up-regulated,and negative regulator Sb SHORT-ROOT1,and Sb RGL1 was down-regulated in GsNAC2 transgenic sorghum after saline-alkali stress.In GsNAC2 transgenic sorghum,receptor genes Sb TIFY9,and Sb TIFY11e,positive regulator Sb JAR1 were up-regulated,and negative regulator Sbb HLH61 were down-regulated.Eighteen differential genes were enriched in glutathione metabolic pathway,the key genes GCL,GS,GSH-Px,and GR in GsNAC2 transgenic sorghum were up-regulated after saline-alkali stress,and all expressed significantly at 24 h;GSH-Px,GR activity,and the content of reduced glutathione（GSH）was also significantly higher than that of the control group.To sum up,GsNAC2 gene plays an important role in the response of sorghum to saline-alkali stress.It can improve the tolerance of transgenic sorghum to saline-alkali stress by promoting the growth,and development of sorghum lines,enhancing the scavenging ability of reactive oxygen species,participating in plant hormone signaling pathway,regulating glutathione metabolism pathway,and promoting reducing glutathione synthesis.