Cloning And Functional Analysis Of UDPGTs Gene In Iris Sanguinea

Cultivating new varieties with new flower colors or with stronger resistance is the main goal of genetic engineering breeding.UDP-glucosyltransferase family genes(UDPGT,UGT)are associated with anthocyanin biosynthesis and are involved in plant defense responses to biological and abiotic stresses,mining UGT gene resources of different plants can lay a foundation for plant genetic engineering breeding.Iris sanguinea is an Iris plant that has both ornamental and medicinal value and is widely used in gardens.In this study,the IsUDPGT gene of I.sanguinea was cloned and analyzed by bio informatics,expression characteristics and subcellular localization,explore the growth of yeast transfected with IsUDPGT gene under different stress treatments,as well as the phenotypic and other changes of transgenic tobacco and Arabidopsis thaliana treated with cadmium chloride,to study the function of gene.The main results are as following:(1)Based on the sequencing of the transcriptome of the Iris,the ORF region of the IsUDPGT gene was cloned by RT-PCR.Bioinformatics analysis shows that,the protein encoded by the nucleotides of the ORF region belongs to the UDP-glycosyltransferase superfamily,encoding 465 amino acids,the protein is an unstable hydrophobic protein without transmembrane helix region,there is a conserved glycosyl receptor binding region pspgbox(342nd-385th)at the c-terminal.Phylogenetic tree analysis showed that the protein encoded by IsUDPGT gene had higher homology with Hevea brasiliensis,followed by Populus euphratica and Rosa chinensis,sequence alignment and homology analysis suggested that the gene belonged to the 3GT subfamily.(2)IsUDPGT gene has different expression levels in flowers and leaves of I.sanguinea and I.sanguinea f.albiflora.In different flowering stages,the expression level of IsUDPGT gene in flowers and leaves of I.sanguinea and I.sanguinea f.albiflora was gradually decreased from SI(bud stage)to S4 stage(decay period).A comparison of the same organization at the same time found that,from S1(bud stage)to S3(full bloom stage),the expression of IsUDPGT gene in petals of I.sanguinea f.albiflora was higher than that in petals of I.sanguinea,but the reverse was found in S4 stage(decay period).At different flowering stages,the expression of IsUDPGT gene in the leaves of I.sanguinea f.albiflora was higher than that of I.sanguinea.The results of subcellular localization showed that the IsUDPGT gene was localized in the nucleus.(3)pYES2-IsUDPGT expression vector was constructed and successfully transferred into yeast strain.Under different concentrations of D-Sorbitol stress,the yeast strain transfected with pYES2-IsUDPGT gene and the yeast strain transfected with vector pYES2 had no significant difference in growth.With the increase of NaCl stress concentration,the two yeasts reacted to the stress,but the difference was not obvious.Under the CdCl2 stress of 150?M/L or above,the growth of the yeast transformed into the pYES2-IsUDPGT gene was significantly weaker than that of the yeast transferred into the pYES2 empty vector,indicating that the transgenic yeast was more sensitive to the stress of cadmium chloride.(4)The plant expression vector pBI121-IsUDPGT-GFP was constructed and transformed into tobacco by Agrobacterium mediated method the IsUDPGT gene was successfully integrated into the tobacco chromosome and expressed.There is no difference in flower color between transgenic tobacco and wild type tobacco.Under different concentrations of CdCl2 treatment,the chlorophyll content in the leaves of transgenic and wild type lines decreased with the increase of CdCl2 concentration,and the yellowing phenomenon of the leaves was gradually obvious.When the stress concentration was 55 ?M/L,110 ?M/L and 275 ?M/L,the leaves state and chlorophyll content of transgenic lines were lower than those of wild type plants,which indicated that transgenic tobacco was sensitive to cadmium stress and weak tolerant to cadmium stress.When the stress concentration reached 550 ?M/L,the degree of injury was the same.(5)The pCXSN-IsUDPGT plant expression vector was constructed,and the Arabidopsis thaliana strain was transformed by inflorescence infection,the IsUDPGT gene was successfully integrated into the Arabidopsis chromosome and expressed.Transgenic and wild type Arabidopsis thaliana during germination were treated with different concentrations of CdCl2,the results showed that the germination rate decreased with the increase of stress concentration,and the plant growth tended to decrease.Under the same stress,the germination rate of transgenic Arabidopsis thaliana seeds was lower than that of wild type plants,the growth of plants was weaker than that of wild type plants,and the fresh weight and root length of transgenic Arabidopsis thaliana plants were lower than those of wild type plants under the same treatment concentration.It is believed that the IsUDPGT gene exhibits a negative regulatory mechanism in the regulation of cadmium resistance.