Study On Key Functional Genes And Mechanism Of Salt Tolerance In Spartina Alterniflora

Spartina alterniflora,originated in North America and South America along the Atlantic coast,has been introducted to many countries in the world.It has become the world main biological invasion ecology and genetics spieces with high reproductive coefficient,strong gene infiltration ability and high resistance to adversity,which is considered to be a model plant to explore salt tolerance genes.Under salt stress,the halophytes can accumulate proline,increase the concentration of cell fluid,reduce osmotic potential,and improve salt tolerance ability.Extensive studies have described the effects of Spartina alterniflora on soil physical,chemical properties and biocommunities,uptake of metal substances,etc.But no detailed studies have focused on the molecular mechanism of proline accumulation in Spartina alterniflora seedlings under salt stress.In this study,Spartina alterniflora was sampled from the salt marsh area of Qingdao,and given different salinity stresses.According to the physiological characteristics of proline accumulation in different tissues,differentially expressed genes in the proline metabolic pathway were screened based on transcriptome sequence technology.The full length of SaP5CS,Sa OAT and SaPro T genes were obtained by RACE technology and bioinformatics analysis was conducted.The expression level of proline metabolism-related genes in different salinities and tissues were elucidated by q RT-PCR.The prokaryotic expression systems of SaP5CS1 and SaP5CS2 were constructed and the spot assay of SaP5CS2protein was performed.The overexpression vector p BWA(V)HS-PC1300-P5CS2 of SaP5CS2,a key gene of proline synthase,was successfully constructed,and the transgenic SaP5CS2 Arabidopsis thaliana T3 seedlings were obtained by Agrobacterium tumefaciens infection.The results were as follows:1)Under salinity stress 24‰at 12 h,the content of proline was significantly different,and the accumulation increased with the increase salt stress.The accumulation of proline in leaves was significantly higher than that in roots and stems.At the same time,chlorophyll content decreased significantly with the increase of stress time,which affected the physiology of Spartina alterniflora.2)Through DEGseq analysis,set the condition by | log₂Fold Change |>1,and q Value<0.05,SaP5CS2 has obvious difference in proline synthesis pathway.3)The full length of SaP5CS1,SaP5CS2,Sa OAT and SaPro T of proline metabolism-related genes in Spartina alterniflora were cloned and analyzed by bioinformatics.The ORF of SaP5CS1,SaP5CS2 were 2,106bp,2,187 bp,encoding 701,728 amino acids.The ORF of Sa OAT was 1,398 bp,encoding 465 amino acids.The ORF of SaPro T was 1,305 bp,encoding 434 amino acids.4)The expression patterns indicated that the accumulation of proline in Spartina alterniflora seedling leaves was mainly due to the SaP5CS2 gene,which regulated glutamate synthesis pathway,playing a more important role under salt stress and promoting proline biosynthesis.5)After low temperature induction and dot plate experiment,the target proteins of SaP5CS1 and SaP5CS2 genes were expressed in Transetta(DE3).With the increase of Na Cl concentration,the density of SaP5CS2protein in LB plate gradually decreased,which preliminarily indicated that SaP5CS2gene is the key salt-tolerance functional gene of Spartina alterniflora.6)The transgenic SaP5CS2 Arabidopsis thaliana T3 seedlings were successfully obtained and treated 10,24 and 32‰seawater for 7 days,respectively.The results showed that the growth status and accumulation of proline of transgenic SaP5CS2 Arabidopsis were significantly higher than that of wild-type,while the accumulation of MDA and H₂O₂were lower than that of wild-type,indicating that overexpression SaP5CS2 gene has been successfully transferred into Arabidopsis,which played an important role in its resistance to salt stress.In conclusion,this study revealed the molecular of proline metabolism and key salt-tolerance functional genes of Spartina alterniflora in response to salt stress,laying a foundation for the study of biological invasion mechanism of Spartina alterniflora.