Sugar Beet M14 Strain Bvm14 - Glyoxalase (i) Gene Cloning And Function Analysis

Monosomic addition line M14is an interspecific hybrid crossed between a wildspecies B. corolliflora Zoss. and a cultivated species B.vulgaris L., which contained thealien chromosome9thof B. corolliflora Zoss. added to the B.vulgaris L. genome. Somereports showed that M14line had exhibited the characteristics of apomixis and stresstolerance (such as drought, high salt and cold), due to the9th chromosome. Therefore,sugar beet M14line has been used as a valuable genetic resource for isolating genesfrom the wild species for improvement in cultivated beet.Previously, comparative proteomics between the flower of M14line and B.vulgarisL. had been performed and71differentially expressed protein spots had beensuccessfully identified. One of the proteins, glyoxalase I is involved in energymetabolism and detoxification of methylglyoxal (MG). The protein level was found toshow a two-fold increase in leaves when the M14plants were treated with salt,indicating that glyoxalase I might involve in the salt-tolerance mechanism in sugar beetM14line. The cloning and function of glyoxalase I from sugar beet M14line wasstudied in the paper.Glyoxalase I is the protein that involve in glycolysis and the TCA cycle, which isubiquitous in animals, plants and microbes. Using reduced glutathione as a cofactor,glyoxalase I catalyzes MG to form S-D-lactoylglutathione (S-LG). And then, S-LG isfurther metabolized to D-lactate and GSH by glyoxalase II. These reactions canmaintain MG levels and decrease its cytotoxicity. Moreover, glyoxalase I had beenproposed to be involved in regulation of cell division and proliferation, microtubularassembly, vesicle mobilization, and had a close relationship with mitosis. Recently,some reports demonstrated that glyoxalase I could be up-regulated by abiotic stresses(such as mannitol, salt, osmotic, etc.) and biotic stresses (for example, Fusarium graminearum), which showed its potent application in crop improvements and thestress-tolerance mechanism.In this study, a cDNA clone, named BvM14-glyoxalase I, was obtained by in silicocloning and RACE strategy. The full-length cDNA was1449bp, which consisted of a1065bp open reading frame and encoded a354aa protein. Sequence analysis showedthere were two glyoxalase domains (93aa-213aa and223aa-343aa) and some other GSHand metal binding sites. Semi-quantitative RT-PCR showed that BvM14-glyoxalase Iwas ubiquitously expressed in different M14tissues, with a high level in flower,followed by stem, root and leaf. The gene could also response to high-salt, mannitol andoxidative stresses, and involved diverse defense mechanisms in the roots and leaves ofsugar beet M14line.To test whether the isolated BvM14-glyoxalase I codes for a functional protein, arecombinant plasmid pET28a-BvM14-glyoxalase I had been constructed, andtransformed into E. coli BL21(DE3). Following induction with0.5mmol/L IPTG and37℃, an additional45kDa fusion protein (greater than the predicted mass39.25kDa)was detected, mostly in the inclusion body of E. coli transformed withpET28a-BvM14-glyoxalase I, but not the E. coli transformed with pET28a vector.Western blotting showed that the fusion protein could be produced correctly. Under thenormal condition, both of E. coli transformed with recombinant plasmids or controlgrew indistinguishably. While in the presence of1mmol/L MG, the growth of bothtransformants was inhibited for9h, followed by early growth ofpET28a-BvM14-glyoxalase I cells and a1h delay for the control cells to grow,indicating the increase tolerance to MG toxicity.A binary vector pBI121-BvM14-glyoxalase I had been constructed for tobaccotransformation. The positive transgenic plants were selected by genomic PCR andRT-PCR. Glyoxalase I activities in two lines (G2and G3) were2.2-fold and1.5-foldhigher than wild-type control plants, indicating successful expression of BvM14-glyoxalase I in tobacco. Leaf disc assay showed that the transgenic plantsmaintained higher chlorophyll contents and delayed chorosis in the leaf discs under thetreatment of various concentrations of MG, NaCl, mannitol and H2O2. The T1generationseedlings line2，derived from G2, were also treated with above stressors and resultsshowed that the survival rate, fresh weight and seedling length of the transgenicseedlings outperformed wild-type plants under the abiotic stresses, especially droughtand oxidative stresses. Taken together, these results indicated that the potent applicationof using BvM14-glyoxalase I to improve salt, drought and oxidative tolerance in crop.This experiment on the gene cloning and function identification ofBvM14-glyoxalase I will lay a foundation for exploring the stress-mechanism ofglyoxalase I.