Physiological Response Of Reaumuria Trigyna To Different Abiotia Stresses And Cloning And Functional Analysis Of RtF3'H1

Reaumuria trigyna belongs to the Reaumuria Linn in the family Tamarieaceae,distributing to the East Alxa-West Ordos in Central Asia,as a recretohalophyte with strong drought tolerance,it is of great significance to desert areas.During the long-term evolution,it is extremely adaptable to the saline-alkali and drought environments.It is significant to explore the response mechanism of R.trigyna to environmental stress.This thesis takes R.trigyna as the research object,from the physiological and molecular levels to carry out the research.The changes of physiological and biochemical indices of R.trigyna were detected and the physiological response mechanism of R.trigyna was investigated under abiotic stresses such as neutral salts NaCl,?NaCl:Na₂SO₄=9:1?,basic salts?NaHCO₃:Na₂CO₃,=9:1?,10%PEG,and UV-B.Based on the transcriptome data,the flavonoid3'-hydroxylase RtF3'H1 gene,a key enzyme gene in the flavonoid synthesis pathway,was cloned from R.trigyna,the expression characteristics and subcellular localization were analyzed.The eukaryotic expression vector of RtF3'H1 gene was constructed and overexpress in Arabidopsis thaliana,then the function of the RtF3'H1 gene and the role of flavonoids under abiotic stresses was investigated.The results will lay a solid foundationan for in-depth understanding of the resistance mechanism of R.trigyna,exploring the molecular mechanism of flavonoid synthesis in wild plants,and discovering resistance-related genes.The main results are as follows:1.The treatment of 10%PEG and UV-B had a greater impact on the growth indexes of the R.trigyna seedlings,followed by alkaline salt,and neutral salt had less effect on the R.trigyna seedlings,it is demonstrated that the R.trigyna had higher tolerance to neutral salt stress.2.Drought,UV-B and alkaline salts cause greater oxidative damage and ionic stress to the R.trigyna seedlings,while the neutral salt has less impact on the R.trigyna seedlings.3.Based on the transcriptome data,the ORF of RtF3'H1 was cloned,with a length of 1512 bp.Bioinformatics analysis revealed that it encodes 504 amino acids,molecular weight is approximately 56.50 kDa.The theoretical isoelectric point is 8.97.The amino acid sequence alignment show that the RtF3'H1 protein has the highest homology with the Catharanthus roseus F3'H,Phylogenetic tree analysis showed that RtF3'H1 was closely related to the F3'H of Glycine max,Ipomoea batatas,Petunia hybrida and Solanum lycopersicum.4.Gene expression characteristics analysis showed that RtF3'H1 was expressed in the roots,stems and leaves of the R.trigyna,with the highest expression in the roots;Different abiotic stresses such as NaCl,PEG,ABA,4?and H₂O₂ can induce the expression of RtF3'H1;And the subcellular localization show that the protein may be localized on the cell membrane and multiple intracellular organelles.5.The pPZP221-RtF3'H1 eukaryotic expression vector was constructed to transform Arabidopsis thaliana to obtain T3 generation transgenic lines.Under NaCl,drought,ABA,and UV-B stress,the functional verification results showed that the growth indexes such as root length,fresh weight,Chlorophyll content were significantly higher than wild type,antioxidant enzyme activities?SOD,POD,CAT?and total flavonoids content were significantly higher than the wild type in the antioxidant enzyme system,anthocyanins content were significantly lower than the wild type.At the same time,H₂O₂ and MDA content were significantly lower than the wild type,DAB and NBT staining show that,less ROS was accumulated in the transgenic lines,the content of osmotic adjustment substance proline was significantly higher than that of wild type,betaine and soluble sugar did not significantly change before and after stress.The above results indicated that overexpression of the RtF3'H1 gene enhances it tolerance to abiotic stress by enhancing the ability of active oxygen scavenging and osmotic regulation of the transgenic Arabidopsis.6.qRT-PCR was used to detect the expression of stress-related genes in RtF3'H1transgenic Arabidopsis thaliana under salt,drought,UV-B and ABA stress,and found that the expression levels of antioxidant enzyme-related genes AtSOD1,AtPOD1,AtCAT1,AtAPX1,stress response genes AtDREB2A,ion transport-related genes AtSOS1,ATNHX1,and proline synthesis-related genes ATP5CS1 in transgenic Arabidopsis were significantly induced.These results indicates that the over-expression of RtF3'H1 gene may regulate the physiological and biochemical response process of plants under abiotic stress by regulating the transcription level of stress-related genes and improve the tolerance of transgenic plants to salt,drought,UV-B and ABA stress.