The amino acid sequence, hydrophilic structure and protein structure were ... oxidase (GO) in E. coli and the application of GO in the production of glyoxylic acid.
Cloning and Sequence Analysis of Glycolate Oxidase Homologues Gene (LcGOX2) from Litchi Pericarp G.B. Hu*, Z.Y. Yang, H.C. Wang, Y.H. Qin and R. Ouyang College of Horticulture South China Agricultural University Guangzhou 510642 China *Corresponding author Keywords: litchi pericarp, DDRT-PCR, glycolate oxidase (GOX), gene cloning, sequence analysis Abstract A full cDNA sequence encoding glycolate oxidase (GOX) was first isolated from litchi pericarp using DDRT-PCR, which was named LcGOX2. The full-length cDNA of LcGOX2 was 1104 bp, encoding 367 amino acids. Sequence analysis showed that LcGOX2 had a high homology in amino acid sequence to previously recorded plant GOX genes and shared the highest (99%) identity with Arabidopsis GOX2. Phylogenetic analysis based on more than 30 GOX amino acid sequences available using DNAstar software suggested that LcGOX2 sequence was the most close to Arabidopsis GOX2 gene. The calculated molecular weight, amino acid composition, hydrophilic and protein structure were also predicted using protein analysis software online (http://cubic.bioc.colubia.edu) and BioEdit software. INTRODUCTION Glycolate oxidase (EC1.3.1.1, GOX) is a key enzyme that catalyzes the oxidation of photosynthetic glycolate to yield glyoxylate and H2O2 in plant leaves. It has been reported that inhibiting GOX activity could significantly reduce C3 plant photorespiration resulting in higher photosynthetic efficiency (Zelitch, 1966, 1975; Zelitch, 1975; Oliver and Zelitch, 1977). Photosynthetic efficiency is an important factor that significant influences crop yield. With the rapid increasing of population and the decreasing arable land in the world, it is necessary to carry out research on how to enhance crop yield through genetic engineering. In recent years, there has been increasing focus on the research of on GOX gene cloning and sequence analysis, and regulation of its expression (Jin et al., 2003; Xu et al., 2006; Zhou et al., 2006). Different GOX genes have been isolated from different plant species such as spinach (Cederlund et al., 1988; Volokita and Somerville, 1987; Park et al., 1992, 1995), lentil (Gerdes and Kindel, 1986, 1988; Ludt and Kindel, 1990), pumpkins (Tsugeki et al., 1993), tomato (Speirs et al., 1995) and Arabidopsis thaliana (Sato et al., 2000). To further our understanding of GOX gene function in litchi, a full cDNA fragment encoding GOX gene was first isolated from litchi pericarp using DDRT-PCR, which named LcGOX2. Based on gene cloning and sequence analysis, further researches on biological function by Southern blot, Northern blot, and anti-GOX gene expression in tobacco are being carried out. MATERIALS AND METHODS Two litchi cultivars ‘Feizixiao’ (stay-green and poor-colored) and ‘Nuomici’ (degreen and well-colored) were selected as experimental materials. Pericarp from early developmental stage (small pericarp), middle stage (turning-red pericarp) and maturity stage (ripe pericarp) were sampled for mRNA differential display (DDRT-PCR) using random primer B026: 5’-GATCTAAGGC-3’ and anchor primer B027 5’-AAGCTTTTTTTTTTG.-3’. A band of DDRT-PCR production (Fig. 1, red arrow) was selected for sequencing analysis. Sequence and phylogenetic analysis were carried out using BLAST of GenBank (http://www.ncbi.nlm.nih.gov/) and DNAstar software Proc. 3rd IS on Longan, Lychee & Other Fruit Trees in Sapindaceae Family Eds.: Qiu Dongliang et al. Acta Hort. 863, ISHS 2010
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respectively. The amino acid sequence, hydrophilic structure and protein structure were predicted using protein analysis software online (http://cubic.bioc.colubia.edu) and BioEdit software. RESULTS Several bands were obtained from litchi pericarp by DDRT-PCR using primers of B0327 and B0326 (Fig. 1). A 1200 pb band of DDRT-PCR production (Fig. 1, red arrow) was selected for sequence analysis. The result showed that it was a full-length cDNA sequence of glycolate oxidase, which was named LcGOX2. LcGOX2 was 1104 bp in length and included an ORF encoding a putative protein of 367 amino acids with a calculated molecular of 40.4 KD (Fig. 2). The deduced amino acids of LcGOX2 were 78-99% identical to the previously recorded plant GOX genes and shared the highest (99% identity) sequence identity with Arabidopsis thaliana GOX2 (Table 1). Phylogenetic analysis based on GOX amino acid sequences available analyzed using DNAstar software suggested that the LcGOX2 sequence were most close to Arabidopsis thaliana, followed by Brassica napus (Fig. 3). The results indicated a close genetic relation and functional conservation among plant GOX genes. Calculated amino acid composition of LcGOX2 as shown in Figure 4, highest percentage of Ala (10.9%) was observed, followed by Leu (8.99%) as compared to the lowest percentage of Cys (0.27%) (Fig. 4). Hydrophilic and protein structure of LcGOX2 were also predicted using protein analysis software online (http://cubic.bioc.colubia.edu) and BioEdit software (Figs. 5 and 6). DISCUSSION GOX genes have been isolated from several plant species (Cederlund et al., 1988; Volokita and Somerville, 1987; Park et al., 1992, 1995; Gerdes and Kindel, 1986, 1988; Ludt and Kindel, 1990; Tsugeki et al., 1993; Speirs et al., 1995; Sato et al., 2000; Zhou et al., 2006). However, there is still no report on GOX genes from woody plants. In this study, a full-length cDNA of GOX gene was obtained from woody plant of litchi pericarp by DDRT-PCR. The deduced amino acids of LcGOX2 cDNA showed high homology (78-99%) with the other plant GOX gene by DNAstar software analysis (Table 1). High homology suggested that the cloned LcGOX2 gene was correct and GOX gene was conserved in all of the characterized plant. As an initial step in elucidating the function of GOX gene, we reported the cloning and sequence analysis of LcGOX2 from litchi pericarp which laid a sound foundation for further studying the LcGOX2 gene in the future. ACKNOWLEDGEMENTS This study has been supported by NSFC (Project No. 30200188), MOST (Project no. 2006BAD01A17) and Ministry of Agriculture (Project no. nyhyzx08-031 and 2006-G31). Literature Cited Gerdes, H.H. and Kindel, H. 1986. Partial purification and characterization of mRNA encoding glycolate oxidase and catalase. Planta 167:166-174. Gerdes, H.H. and Kindel, H. 1988. Gene expression upon illumination in forming mRNA encoding peroxisomal glycolate oxidase. Biochem Biophys Acta 949:195-205. Jin, J.F., Tan, T.W., Wang, H.L. and Su, G.F. 2003. The expression of spinach glycolate oxidase (GO) in E. coli and the application of GO in the production of glyoxylic acid. Mol. Biotech. 25(3):207-214. Ludt, C. and Kindel, H. 1990. Characterization of a cDNA encoding Lens culinaris glycolate oxidase and developmental expression of glycolate oxidase mRNA in cotyledons and leaves. Plant Physiol. 94:1193-1198. Oliver, D and Zelitch, I. 1977. Increased photosynthesis by inhibition of photorespiration with glyoxylate. Science 196:1450-1451.
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Park, Y.S., Chai, J.D. and Cho, N.J. 1992. Expression of glycolate oxidase gene in spinach. Korean Biochem. 25:219-232. Park, Y.S., Jin, Y.H., Kim, Y.C., Choi, J.D. and Cho, N.J. 1995. Effect of light on spinach glycolate oxidase gene expression. J. Biochem. Mol. Bol. 28(3):217-274. Sato, S., Namur, Y., Kaneko, T., Katoh, T., Asamizu, E. and Tabata, S. 2000. Structural analysis of Arabidopsis thaliana chromosome 3. DNA Res. 7:131-135. Somerville, C.R. and Ogren, W.L. 1980. Inhibition of photosynthesis in mutants of Arabidopsis. Nature 257-286. Tsugeki, R., Hara-Nishimura, I. and Mori, H. 1993. Cloning and sequencing of cDNA for glycolate oxidase from pumpkin cotyledons and Northern blot analysis. Plant Cell Physiol. 34:51-55. Volokita, M. and Somerville, C.R. 1987. The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone. J. Biol. Chem. 262(33):15825-15828. Wang, H.C., Huang, X.M., Hu, G.B., Yang, Z.Y. and Huang, H.B. 2005. A comparative study of chlorophyll loss and its related mechanism during fruit maturation in the pericarp of fast- and slow-degreening litchi pericarp. Scientia Horticulturae 106:247-257. Xu, H.W., Ji, X.M., He, Z.H., Shi, W.P., Zhu, G.H., Niu, J.K., Li, B.S. and Peng, X.X. 2006. Oxalate accumulation and regulation is independent of Oxalate accumulation and regulation is independent of glycolate oxidase in rice leaves. J. Exp. Bot. 57(9):1899-1908. Zelitch, I. 1966. Increased rate of net photosynthetic carbon dioxide uptake caused by the inhibition of glycolate oxidase. Plant Physiol. 41:1623-1631. Zelitch, I. 1975. Plant productivity and control of photorespiration pathways of carbon fixation in green plant. Science 188(4188):625-635. Zhou, S.P., Chen, F.C., Nahashon, S. and Chen, T.T. 2006. Cloning and characterization of glycolate oxidase and NADH-dependent hydropyruvate reductase genes in Pachysandra terminalis. HortScience 41(5):1226-1230.
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Tables
Table 1. Comparison of the deduced amino acid sequence of the LcGOX2 with GOX genes of other plants. Gene GOX2 AT3g14420/MOA2_2 Glycolate oxidase GOX1 Glycolate oxidase Glycolate oxidase GOX-like Glycolate oxidase GOX Glycolate oxidase Glycolate oxidase
Registered no. in GenBank Q9LRR9 AAL16258.1 AAV28535.1 Q9LRSO AAB40396.1
Homology (%) 99 98 95 94 91
BAA03131.1 1AL7 AAO17067.1 PO5414 1803516A AAB82143.1
89 89 88 88 88 78
Plant species Arabidopsis thaliana Arabidopsis thaliana Brassica napus Arabidopsis thaliana Mesembryanthemum crystallinum Cucurbita Spinacia oleracea Zantedeschia aethiopica Spinacia oleracea Lens culinaris Oryza sativa
Figures
Fig. 1. DDRT-PCR product using B0327 and B0326 primers. M, DL2000 Marker; 1: ‘Nuomici’ green pericarp; 2: ‘Nuomici pericarp at color break; 3: ‘Nuomici’ pericarp at ripe stage; 4: ‘Feizixiao’ young and green pericarp; 5: ‘Feizixiao’ pericarp at color break; 6: Red ‘Feizixiao’ pericarp at ripe stage; and 7: Green ‘Feizixiao’ pericarp at ripe stage.
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Fig. 2. Nucleotide and deduced amino acid sequences of the full-length cDNA of LcGOX2 gene from litchi pericarp.
Fig. 3. Phylogenetic relationship of the LcGOX2 with the other plant GOX gene.
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Fig. 4. Calculated amino acid composition of LcGOX2.
Fig. 5. Calculated hydrophilic structure of LcGOX2.
Fig. 6. Calculated protein structure of LcGOX2.
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respectively. The amino acid sequence, hydrophilic structure and protein structure were predicted using protein analysis software online (http://cubic.bioc.colubia.edu) and BioEdit software. RESULTS Several bands were obtained from litchi pericarp by DDRT-PCR using primers of B0327 and B0326 (Fig. 1). A 1200 pb band of DDRT-PCR production (Fig. 1, red arrow) was selected for sequence analysis. The result showed that it was a full-length cDNA sequence of glycolate oxidase, which was named LcGOX2. LcGOX2 was 1104 bp in length and included an ORF encoding a putative protein of 367 amino acids with a calculated molecular of 40.4 KD (Fig. 2). The deduced amino acids of LcGOX2 were 78-99% identical to the previously recorded plant GOX genes and shared the highest (99% identity) sequence identity with Arabidopsis thaliana GOX2 (Table 1). Phylogenetic analysis based on GOX amino acid sequences available analyzed using DNAstar software suggested that the LcGOX2 sequence were most close to Arabidopsis thaliana, followed by Brassica napus (Fig. 3). The results indicated a close genetic relation and functional conservation among plant GOX genes. Calculated amino acid composition of LcGOX2 as shown in Figure 4, highest percentage of Ala (10.9%) was observed, followed by Leu (8.99%) as compared to the lowest percentage of Cys (0.27%) (Fig. 4). Hydrophilic and protein structure of LcGOX2 were also predicted using protein analysis software online (http://cubic.bioc.colubia.edu) and BioEdit software (Figs. 5 and 6). DISCUSSION GOX genes have been isolated from several plant species (Cederlund et al., 1988; Volokita and Somerville, 1987; Park et al., 1992, 1995; Gerdes and Kindel, 1986, 1988; Ludt and Kindel, 1990; Tsugeki et al., 1993; Speirs et al., 1995; Sato et al., 2000; Zhou et al., 2006). However, there is still no report on GOX genes from woody plants. In this study, a full-length cDNA of GOX gene was obtained from woody plant of litchi pericarp by DDRT-PCR. The deduced amino acids of LcGOX2 cDNA showed high homology (78-99%) with the other plant GOX gene by DNAstar software analysis (Table 1). High homology suggested that the cloned LcGOX2 gene was correct and GOX gene was conserved in all of the characterized plant. As an initial step in elucidating the function of GOX gene, we reported the cloning and sequence analysis of LcGOX2 from litchi pericarp which laid a sound foundation for further studying the LcGOX2 gene in the future. ACKNOWLEDGEMENTS This study has been supported by NSFC (Project No. 30200188), MOST (Project no. 2006BAD01A17) and Ministry of Agriculture (Project no. nyhyzx08-031 and 2006-G31). Literature Cited Gerdes, H.H. and Kindel, H. 1986. Partial purification and characterization of mRNA encoding glycolate oxidase and catalase. Planta 167:166-174. Gerdes, H.H. and Kindel, H. 1988. Gene expression upon illumination in forming mRNA encoding peroxisomal glycolate oxidase. Biochem Biophys Acta 949:195-205. Jin, J.F., Tan, T.W., Wang, H.L. and Su, G.F. 2003. The expression of spinach glycolate oxidase (GO) in E. coli and the application of GO in the production of glyoxylic acid. Mol. Biotech. 25(3):207-214. Ludt, C. and Kindel, H. 1990. Characterization of a cDNA encoding Lens culinaris glycolate oxidase and developmental expression of glycolate oxidase mRNA in cotyledons and leaves. Plant Physiol. 94:1193-1198. Oliver, D and Zelitch, I. 1977. Increased photosynthesis by inhibition of photorespiration with glyoxylate. Science 196:1450-1451. 118
Park, Y.S., Chai, J.D. and Cho, N.J. 1992. Expression of glycolate oxidase gene in spinach. Korean Biochem. 25:219-232. Park, Y.S., Jin, Y.H., Kim, Y.C., Choi, J.D. and Cho, N.J. 1995. Effect of light on spinach glycolate oxidase gene expression. J. Biochem. Mol. Bol. 28(3):217-274. Sato, S., Namur, Y., Kaneko, T., Katoh, T., Asamizu, E. and Tabata, S. 2000. Structural analysis of Arabidopsis thaliana chromosome 3. DNA Res. 7:131-135. Somerville, C.R. and Ogren, W.L. 1980. Inhibition of photosynthesis in mutants of Arabidopsis. Nature 257-286. Tsugeki, R., Hara-Nishimura, I. and Mori, H. 1993. Cloning and sequencing of cDNA for glycolate oxidase from pumpkin cotyledons and Northern blot analysis. Plant Cell Physiol. 34:51-55. Volokita, M. and Somerville, C.R. 1987. The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone. J. Biol. Chem. 262(33):15825-15828. Wang, H.C., Huang, X.M., Hu, G.B., Yang, Z.Y. and Huang, H.B. 2005. A comparative study of chlorophyll loss and its related mechanism during fruit maturation in the pericarp of fast- and slow-degreening litchi pericarp. Scientia Horticulturae 106:247-257. Xu, H.W., Ji, X.M., He, Z.H., Shi, W.P., Zhu, G.H., Niu, J.K., Li, B.S. and Peng, X.X. 2006. Oxalate accumulation and regulation is independent of Oxalate accumulation and regulation is independent of glycolate oxidase in rice leaves. J. Exp. Bot. 57(9):1899-1908. Zelitch, I. 1966. Increased rate of net photosynthetic carbon dioxide uptake caused by the inhibition of glycolate oxidase. Plant Physiol. 41:1623-1631. Zelitch, I. 1975. Plant productivity and control of photorespiration pathways of carbon fixation in green plant. Science 188(4188):625-635. Zhou, S.P., Chen, F.C., Nahashon, S. and Chen, T.T. 2006. Cloning and characterization of glycolate oxidase and NADH-dependent hydropyruvate reductase genes in Pachysandra terminalis. HortScience 41(5):1226-1230.
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Tables
Table 1. Comparison of the deduced amino acid sequence of the LcGOX2 with GOX genes of other plants. Gene GOX2 AT3g14420/MOA2_2 Glycolate oxidase GOX1 Glycolate oxidase Glycolate oxidase GOX-like Glycolate oxidase GOX Glycolate oxidase Glycolate oxidase
Registered no. in GenBank Q9LRR9 AAL16258.1 AAV28535.1 Q9LRSO AAB40396.1
Homology (%) 99 98 95 94 91
BAA03131.1 1AL7 AAO17067.1 PO5414 1803516A AAB82143.1
89 89 88 88 88 78
Plant species Arabidopsis thaliana Arabidopsis thaliana Brassica napus Arabidopsis thaliana Mesembryanthemum crystallinum Cucurbita Spinacia oleracea Zantedeschia aethiopica Spinacia oleracea Lens culinaris Oryza sativa
Figures
Fig. 1. DDRT-PCR product using B0327 and B0326 primers. M, DL2000 Marker; 1: ‘Nuomici’ green pericarp; 2: ‘Nuomici pericarp at color break; 3: ‘Nuomici’ pericarp at ripe stage; 4: ‘Feizixiao’ young and green pericarp; 5: ‘Feizixiao’ pericarp at color break; 6: Red ‘Feizixiao’ pericarp at ripe stage; and 7: Green ‘Feizixiao’ pericarp at ripe stage.
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Fig. 2. Nucleotide and deduced amino acid sequences of the full-length cDNA of LcGOX2 gene from litchi pericarp.
Fig. 3. Phylogenetic relationship of the LcGOX2 with the other plant GOX gene.
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Fig. 4. Calculated amino acid composition of LcGOX2.
Fig. 5. Calculated hydrophilic structure of LcGOX2.
Fig. 6. Calculated protein structure of LcGOX2.
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