Cloning And Functional Analysis Of RtLdox2 In Reaumuria Trigyna

Abiotic stresses including salt,drought,UV-B affect the growth and development of plants.Plants can accumulate secondary metabolites to resist different stresses.Anthocyanins have strong antioxidant activities and reduce the damage of reactive oxygen species.Previous studies have shown that anthocyanin plays an important role in environmental resistance of plants.Reaumuria trigyna belongs to Reaumuria Linn in the family Tamariaceae and is endemic to the eastern Alxa-Western Ordos in central Asia.It is highly adaptable to the saline and desert environment.Also it is a suitable material to study the function of plants under different abiotic stress.In this study,the RtLDOX2 gene was cloned from R.trigyna,and the expression characteristics of RtLDOX2 under different stresses were analyzed.We constructed the prokaryotic expression to transform E.coli.Then analysis the catalytic activity of Rt LDOX2 protein.The function of Rt LDOX2 transgenic Arabidopsis under various abiotic stress were detected,The findings have a significant implications for exploring the regulation of anthocyanin synthesis on plant resistance to abiotic stresses.The main results of this study are as follows:?1?.The ORF of Rt LDOX2 gene was cloned from R.trigyna,and the length is1074 bp,encoding 357 amino acids,Molecular weight approximately 39.90 kDa.The theoretical isoelectric point is 5.62.Structural analysis revealed that the RtLDOX2 contains three exons and two introns.The amino acid sequence alignment showed that Rt LDOX2 had two conserved domains.Compared with RtLDOX,there is one more conserved domain of 2-oxoglutarate and Fe²⁺-oxygenase superfamily.Phylogenetic tree results suggested that RtLDOX2 was closely related to the LDOX ofNoccaeacaerulescens,Dichantheliumoligosanthes,Phalaenopsis equestris.However,RtLDOX2 had a distant relationship with RtLDOX.The promoter sequence of the gene was cloned using chromosome walking technique,Analysis of promoter sequence indicated that it had elements response to stress such as G-box,MRE,ABRE,and ERE.?2?.Tissue specificity analysis indicated that Rt LDOX2 was expressed in root,stem and leaves,and the expression level was lower in stem.Different abiotic stresses such as NaCl,PEG,4oC,H₂O₂ and ABA could induce the expression of RtLDOX2.?3?.Constructing the Rt LDOX2 prokaryotic expression vector,and transforming into the expressional strains.Then induces and purifies the protein,Finally,receives the RtLDOX2 recombinant protein.The recombinant protein catalyzes naringenin,dihydrokaempferol,dihydroquercetin,and dihydromyricetin.It was found that RtLDOX2 has the catalytic activity of FLS.?4?.Constructing the RtLDOX2 eukaryotic expression vector,and transforming into Arabidopsis thaliana.Under NaCl,drought,UV-B and sucrose treatment,It found that root length,fresh weight and chlorophyll content of transgenic plants were significantly higher than WT,Compared with WT,proline,activities of antioxidant enzymes?SOD,POD,CAT?and anthocyanin,total flavonoids were higher in transgenic Arabidopsis.The contents of MDA,H₂O₂,and PA were lower.Expression levels of stress-related genes were detected.It was revealed that expression levels of antioxidant-related genes,ion transport-related genes,and proline synthesis-related genes of transgenic plants under stress were notably higher than those of WT.The findings showed that Rt LDOX2 increased the antioxidant and Osmotic capacity of plants by increasing the content of anthocyanins and enhanced the resistance to abiotic stress.