Cloning And Function Analysis Of A D-lactate Dehydrogenase Gene Involved In Methyglyoxal Pathway In Rice

Rice (Oryza sativa L.), one of the most important crops in the world, often encounters flash flooding of fields during growth and development, which could cause complete submergence and result in hypoxia or even anaerobic environment, leading to anaerobic respiration including alcoholic fermentation, lactic acid fermentation and alanine fermentation. Lactic acid fermentation is a biological process by which pyruvate is converted into cellular energy and the metabolite lactate which is catalyzed by lactate dehydrogenases. Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are NAD(P)-dependent enzymes, each acting on either D-lactate (EC1.1.1.28) or L-lactate (EC1.1.1.27), using NAD(P) as proton receptor. The other two are cytochrome c-dependent enzymes, each acting on either D-lactate (EC1.1.2.4) or L-lactate (EC1.1.2.3), using cytochrome c as electron receptors. These four types of lactate dehydrogenases are extensively found in bacteria, Archaebacteria and eukaryotes.This dissertation was focused on cloning and factional characterization of a rice Cytochrome C-dependent D-lactate dehydrogenase (OsD-LDH). The substate screen, electron receptors, optimum pH and temperature and Km Vs Vmax of OsD-LDH were investigated through prokaryotic expression and purification. The expression pattern of OsD-LDH were analysed using Real-Time PCR. Subcellular localization in rice protoplasts revealed that OsD-LDH-GFP accumulated in mitochondria. In order to characterize the function of OsD-LDH, interference transgenic plants were created through Agrobacterium tumefaciens-mediated transformation. The results were shown as follow:1. Gene cloning:OsD-LDH cDNA was cloned from Oryza sativa L. Indica cv.9311through bioinformatics analysis and followed by RT-PCR. The OsD-LDH locus was located on the short arm of chromosome7and this enzyme contained homologous regions to a FAD-binding domain and a C-terminal FAD-oxidase domain. Several alternative splicing types of OsD-LDH were obtained after cDNA sequencing, including two main types named as OsD-LDH-1and OsD-LDH-2, which encoded254amino acids and561amino acids, respectively. There were additional7bp in the coding domain sequence of OsD-LDH-1, where a premature termination codon was introduced to result in the truncated protein;2. Analysis of conservation and evolution:OsD-LDH-2protein shared a79.5%similarity and69.8%identity to AtD-LDH, a25.3%similarity and18.2%identity to ScDLDH and a20.5%similarity and13.7%identity to EcDLDH. Phylogenetic analysis revealed that CytC-dependent D-LDHs were widespread across prokaryotes and eukaryotes;3. Analysis of enzyme activities and kinetic parameters:OsD-LDH-2-GST protein was purified through prokaryotic expression and GST resin affinity purification. The characterization of enzyme activities revealed that OsD-LDH-2-GST could catalyze D-Lactate into pyruvate using CytC as electron receptors. The highest activity was found with D-2-hydroxybutyrate as substrate, with a50%higher Vmax and a50%lower Km than D-Lactate. OsD-LDH-2-GST could also catalyze L-Lactate into pyruvate, with about10%Vmax of D-Lactate. Glycolate was not the substrate of OsD-LDH-2-GST; OsD-LDH-2overall was a High affinity and moderate efficiency enzyme, whereas OsD-LDH-1almost lost whole activities;4. Subcellular localization analysis:OsD-LDH-GFP accumulated in mitochondria with a signal peptide in the first40amino acids of the N-terminal; 5. Tissue-specific expression analysis:The expression pattern of OsD-LDH was analysed from various tissues at different development stages of9311using Real-Time PCR. As a result, OsD-LDH mRNA accumulated to high levels in flag leaves, to moderate levels in flag leaf sheaths and germinated seeds and to relatively low level in all the other tissues. It should be noticed that the expression levels of OsD-LDH gradually increased during the1-10DAF. And the expression pattern of OsD-LDH was very different from NAD-dependent OsL-LDH; Further analysis of the response to abiotic stress and hormone treatment by qRT-PCR revealed that expressions of OsD-LDH were elevated in heat shock and GA, SA, IAA and6-BA treatments, which was also different from NAD-dependent OsL-LDH.6. Creating interference transgenic plants:OsD-LDH RNAi construct was introduced into Oryza sativa L. japonica cv. ZH11by Agrobacterium tumefaciens-mediated transformation to obtain33independent transgenic lines.30positive lines were verified by genomic PCR, among which, the expression levels of OsD-LDH were declined in12transgenic lines tested by RT-PCR.7. Functional analysis:RNAi transgenic plants showed severely growth retardation under the treatment of methyglyoxal compared to wild type and control. Further analysi revealed that the methyglyoxal was accumulated in high level, the total glutathione level decreased with severely decreased GSH:GSSG ratio and the activities of glyoxalase system were altered in RNAi transgenic plants, which indicated that OsD-LDH was involved in methyglyoxal metabolism through clearing D-Lactate produced from glyoxalase system. Additionally, OsD-LDH was also involved in response to NaCl stress.