Cloning And Functional Analysis Of Potential Salt-Tolerant Genes Of GDH2 And NACs In Industrial Hemp (Cannabis Sativa L.)

Salt stress is a major abiotic stress that affects plant growth and development and the global agricpltural production.Therefore,research on the salt tolerance mechanism of plants has become one of the hotspots of global concern.Industrial hemp {Cannabis sativa L.)is a very prorrusing economic crop.It is of great theoretical and practical significance to study the salt tolerance mechanism and improve the salt tolerance of hemp.At present.NAC transcription factor have been confirmed to response to salt stress,and glutamate deaminase(GDH)is an important stress-responsive enzyme in plants.Based on the preliminary work of exploring the hemp tissue culture system,four candidated genes were screened from the transcriptome database of hemp,named CsGDH2,CsNACL CsNAC2,and CsNAC3,all of which were confirmed to participate in the stress response pathway.In this paper,the f?ll-length sequences of four genes were cloned for bioinformatics analysis.The expression of the four genes was analyzed by screening the internal reference senes.The prokaryotic expression vector and RNAi and overexpression vectors were constructed,and the overexpression vectors were transformed into tobacco and Arabidopsis thaliana to verify the function of these four salt stress response genes;the main res?lts are as follows:(1)RT-PCR and RT-qPCR analysis indicated that these four genes are expressed in roots and leaves,and the expression levels varied with salt concentration and stress time.The expression level of CsGDH2 gene in leaves was higher than that in roots.Under 200 mM NaCI stress,the expression level of CsA4C1 gene in roots was higher than that in leaves,while CsNAC2 and CsNAC3 genes were opposite.In addition,the expression levels of CsGDH2 and CsNAC3 genes increased with the increase of stress time,but CsNA C1 and CsNA C2 showed a trend of increasing firstly and then decreasing.(2)The GDH2 and NAC genes from hemp were successf?lly constructed into the prokaryotic expression vector of pEASY-Blunt E1,and a large number of target proteins were induced under salt stress,indicating that four genes may be involved in the regulation of salt stress of hemp(3)The cDNA fragment of glutamate dehydrogenase 2 gene(CsGDH2)was successf?lly cloned from hemp.The f?ll length of cDNA fragment was 1730 bp and the open reading frame was 1236 bp,encoding 411 amino acids,including a conserved domain of glutamate dehydrogenase.Phylogenetic tree analysis found that CsGDH2 has the closest relationship with Morus notabilis,and the relationship with cucubita and momordica was the farthest.The full-length cDNA of CsNAC1,CsNAC2 and CsNAC3 were 888 bp,864 bp and 1059 bp,encoding 295.287 and 352 amino acids,respectively.It was found that their sequences contains NAM domains,in addtion.CsNAC1 and CsNAC2 belong to the ATAF subfamily of the NAC family,and CsNAC3 belongs to the AtNAC3 subfamily.Phylogenetic tree showed that they are related to ramie,parasponia andersonii and ricinus communis.In addition,the physicochemical properties(isoelectric point,fat solubility coefficient,instability coefficient,etc)of the hydrophilic proteins encoded by the above four genes were briefly analyzed(4)The RNAi and overexpression vectors of these four genes were constructed and transferred into tobacco and Arabidopsis thaliana by the method of leaf disc and inflorescence infection:? The GDH activity.SOD activity,chlorophyll content and soluble sugar content of CsGDH2 transgenic tobacco were significantly higher than wild type,and the MD A content was significantly lower than that of wild type.? The relative conductivity and SOD activity of CsNAC1 and CsNAC3 transgenic tobacco were significantly higher than those of wild type.and these two physiological indexes of CsNAC2 transgenic tobacco were not significantly difference to wild type.The chlorophyll content and soluble sugar content were significantly higher than wild type;the MDA content of CsNAC1 and CsNAC3 transgenic plants was significantly lower than that of wild tvpe.but there was no significant difference in CsNAC2 transgenic plants.? The gerrnination rate of CsGDH2,CsNAC2 and CsNAC3(CsNAC1 gene infection failed)transgenic Arabidopsis seeds under salt stress was significantly higher than that of wild type,indicating that salt tolerance was improved.In summary,overexpression of CsGDH2,CsNAC1 and CsNAC3 can significantly improve the salt tolerance of transgenic tobacco,and the function of CsNAC2 gene needs to be further determined(5)For "Yunma l ' cultivar,the optimal culture medium for leaf callus induction was 0.4 mg/L TDZ+0.1 mg/L TAA+MS+8g Agar+30g Sugar;The selection pressure of hygromycin for leaf callus induction was 2 mg/L,and that of cephalosporin was 0.2 g/L.