Overexpression Of A Tomato UDP-glucosytransferase Gene Enhance Chilling Tolerance In Tobacco

Plants will encounter with many environment stresses in their growth process which can seriously affect their yields and quality. Meanwhile, a change of osmotic regulation substances, membrane fatty acid composition caused by the expression of some relevant genesinthe plants. Some other genes whereas also can enhance stress tolerant through changing the level of some tolerant substance, such as transcription factors, glycotransferases and so on. More and more attentions have been paid to the function of glycotransferase, because of the effect of increasing stress resistance in directly by changing the plant metabolites, endogenous hydrophilic substances, structural stability, subcellular localization, biological activity.In this study, a glycotransferase gene named LeUGT73C5 specific glycosylation of BR was isolated and characterized from tomato leaf. The main results are as follows:(1) The full length sequence of LeUGT73C5 was cloned from the tomato leaf treated under low temperature using RT-PCR and the primer designed on the basis of NCBI. The gene contains 1735 bp nucleotides with an open reading frame (ORF) of 1485 bp comprising 494 amino acid residues with the predicted molecular mass of 54 kDa. The deduced amino acid sequence showed high identities with UGT73C5 from Ricinuscommunis, Vitisvinifera, Populustrichocarpa, Oryzasativa, Arabidopsis thaliana. Amino acid sequence alignment revealed that the isolated cDNA contained the previously defined common domain, the PSPG box.(2) p35S-LeUGT73C5-GFP fusion protein was constructed and transiently expressed in Arabidopsis protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating the LeUGT73C5 subcellular localization on chloroplast.(3) Northern hybridization showed that the transcript of LeUGT73C5 exited in all the detected tissues including flowers, stems, leaf and roots, but with an abundant expression level in the leaf. The expression of LeUGT73C5 was obviously induced by low temperature and MV treatment.(4) The full length LeUGT73C5 cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and the transgenic tabacco plants were verified by PCR and northern hybridization. It was indicated that the LeUGT73C5 gene had been recombined into tabacco genome and the sense transgenic tobacco plants were obtained.(5) A recombinant of prokaryotic expression vector pET- LeUGT73C5 was constructed and transformed to E.Coli BL21. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. Western hybridization revealed the presence of a strong positive protein signal corresponding to LeUGT73C5 in sense transgenic plants.(6) The growth analysis of WT and transgenic tobacco indicated that under normal conditions there is no difference in the growth of all plants. But under chilling stress, the transgenic tobacco lines showed more adaptive characteristic than WT plants, especially the recovery after the stress.(7) Although net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) in WT and transgenic plants decreased markedly under chilling stress in the low irradiance (4℃, 100μmolm^-2s^-1), the decrease of Pn and Fv/Fm was more slower in sense transgenic plants than in WT. The results indicated that overexpression of LeUGT73C5 alleviated the photoinhibition of PSII. Compared to WT, the sense-transgenic lines could maintain higher activities of chloroplastic SOD and APX, lower content of chloroplastic O₂(?) and H₂O₂ under chilling stress.