Effective Exon-Intron Structure Verification of a 1-Pyrroline-5-Carboxylate-Synthetase Gene from Halophytic Leymus chinensis (Trin.) Based on PCR, DNA Sequencing, and Alignment

  • Sun, Yan-Lin (Department of Bio-Health Technology, College of Biomedical Science, Kangwon National University) ;
  • Hong, Soon-Kwan (Department of Bio-Health Technology, College of Biomedical Science, Kangwon National University)
  • Received : 2010.06.22
  • Accepted : 2010.09.27
  • Published : 2010.12.31

Abstract

Genomes of clusters of related eukaryotes are now being sequenced at an increasing rate. In this paper, we developed an accurate, low-cost method for annotation of gene prediction and exon-intron structure. The gene prediction was adapted for delta 1-pyrroline-5-carboxylate-synthetase (p5cs) gene from China wild-type of the halophytic Leymus chinensis (Trin.), naturally adapted to highly-alkali soils. Due to complex adaptive mechanisms in halophytes, more attentions are being paid on the regulatory elements of stress adaptation in halophytes. P5CS encodes delta 1-pyrroline-5-carboxylate-synthetase, a key regulatory enzyme involved in the biosynthesis of proline, that has direct correlation with proline accumulation in vivo and positive relationship with stress tolerance. Using analysis of reverse transcription-polymerase chain reaction (RT-PCR) and PCR, and direct sequencing, 1076 base pairs (bp) of cDNA in length and 2396 bp of genomic DNA in length were obtained from direct sequencing results. Through gene prediction and exon-intron structure verification, the full-length of cDNA sequence was divided into eight parts, with seven parts of intron insertion. The average lengths of determinated coding regions and non-coding regions were 154.17 bp and 188.57 bp, respectively. Nearly all splice sites displayed GT as the donor sites at the 5' end of intron region, and 71.43% displayed AG as the acceptor sites at the 3' end of intron region. We conclude that this method is a cost-effective way for obtaining an experimentally verified genome annotation.

Acknowledgement

Supported by : Nutraceutical Bio Brain Korea 21 Project Group

References

  1. Fabro, G., I. Kovács, V. Pavet, L. Szabados and M.E. Alvarez. 2004. Proline accumulation and AtP5CS2 gene activation are induced by plant-pathogen incompatible interactions in Arabidopsis. MPMI. 17: 343-350. https://doi.org/10.1094/MPMI.2004.17.4.343
  2. Fickett, J.W. and C.S. Tung. 1992. Assessment of protein coding measures. Nucleic Acids Res. 20: 6441-6450. https://doi.org/10.1093/nar/20.24.6441
  3. Fujita, T., A. Maggio, M. Garcia Rios, R.A. Bressan and L.N. Csonka. 1998. Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for $\Delta^{1}$-pyrroline-5-carboxylate synthetase from tomato. Plant Physiol. 118: 661-674. https://doi.org/10.1104/pp.118.2.661
  4. Ginzberg, I., H. Stein, Y. Kapulnik, L. Szabados, N. Strizhov, J. Schell, C. Koncz and A. Zilberstein. 1998. Isolation and characterization of two different cDNAs of $\Delta^{1}$-pyrroline- 5-carboxylate synthetase in alfalfa, transcriptionally induced upon salt stress. Plant Nol. Biol. 38: 755-764. https://doi.org/10.1023/A:1006015212391
  5. Hamilton, E.W. and S.A. Heckathorn. 2001. Mitochondrial adaptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiol. 126: 1266-1274. https://doi.org/10.1104/pp.126.3.1266
  6. Hoagland, D.R. and D.I. Arnon. 1950. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 347: 1-32.
  7. Hoekstra, F.A., E.A. Golovina and J. Buitink. 2001. Mechanisms of plant desiccation tolerance. Trends Plant Sci. 6: 431-438. https://doi.org/10.1016/S1360-1385(01)02052-0
  8. Huang, Z., J. Zhu, X. Mu and J. Jin. 2004. Pollen dispersion, pollen viability and pistil receptivity in Leymus chinensis. Ann. Bot. 93: 295-301. https://doi.org/10.1093/aob/mch044
  9. Mironov, A.A., J.W. Ficket and M.S. Gelfand. 1999. Frequent alternative splicing of human genomes. Genome Res. 9: 1288-1293. https://doi.org/10.1101/gr.9.12.1288
  10. Oliver, S.G., Q.J.M. Van Der Aart, M.L. Agostoni Carbone, M. Aigle, L. Alberghina, D. Alexandraki, G. Antoine, R. Anwar, J.P.G. Ballesta, P. Benit, et al. 1992. The complete DNA sequence of yeast chromosome III. Nature. 357: 38-46. https://doi.org/10.1038/357038a0
  11. Pollard, A. and R.G. Wyn Jones. 1979. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta. 144: 291-298. https://doi.org/10.1007/BF00388772
  12. Rhodes, D., S. Handa and R.A. Bressan. 1986. Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol. 82: 890-903. https://doi.org/10.1104/pp.82.4.890
  13. Rogic, S., A.K. Mackworth and F.B.F. Ouellett. 2001. Evaluation of gene-finding programs on mammalian sequences. Genome Res. 11: 817-832. https://doi.org/10.1101/gr.147901
  14. Roosens, N.H., R. Willem, Y. Li, I. Verbruggen, M. Biesemans and M. Jacobs. 1999. Proline metabolism in the wild-type and in a salt-tolerant mutant of Nicotiana plumbaginifolia studied by $^{13}C$-nuclear magnetic resonance imaging. Plant Physiol. 121: 1281-1290. https://doi.org/10.1104/pp.121.4.1281
  15. Savoure, A., S. Jaoua, X.J. Hua, W. Ardiles, M. Van Montagu and N. Verbruggen. 1995. Isolation, characterization, and chromosomal location of a gene encoding the $\Delta$1-pyrroline- 5-carboxylate synthetase in Arabidopsis thaliana. FEBS Lett. 372: 13-19. https://doi.org/10.1016/0014-5793(95)00935-3
  16. Schulz, R.A. and B.A. Butler. 1989. Overlapping genes of Drosophila melanogaster: organization of the z600-gonadal Eip28/29 gene cluster. Genes Dev. 3: 232-242. https://doi.org/10.1101/gad.3.2.232
  17. Solovyev, V.V., A.A. Salamov and C.B. Lawrence. 1994. Predicting internal exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames. Nucl. Acids Res. 22: 5156-5163. https://doi.org/10.1093/nar/22.24.5156
  18. Tenney, A.E., R.H. Brown, C. Vaske, J.K. Lodge, T.L. Doering and M.R. Brent. 2004. Gene prediction and verification in a compact genome with numerous small introns. Genome Res. 14: 2330-2335. https://doi.org/10.1101/gr.2816704
  19. Wilson, R., R. Ainscough, K. Anderson, C. Baynes, M. Berks, J. Bonfield, J. Burton, M. Connell, T. Copsey, J. Cooper, et al. 1994. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 368: 32-38. https://doi.org/10.1038/368032a0
  20. Yoshiba, Y., T. Kiyosue, T. Katagiri, H. Ueda, T. Mizoguchi, K. Yamaguchi Schinozaki, K. Wada, Y. Harada and K. Shinozaki. 1995. Correlation between the induction of a gene for $\Delta^{1}$-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J. 7: 751-760. https://doi.org/10.1046/j.1365-313X.1995.07050751.x
  21. Delauney, A.J. and D.P.S. Verma. 1993. Proline biosynthesis and osmoregulation in plants. Plant J. 4: 215-223. https://doi.org/10.1046/j.1365-313X.1993.04020215.x
  22. Dunham, I., N. Shimizu, B.A. Roe, S. Chissoe, et al. 1999. The DNA sequence of human chromosome 22. Nature. 402: 489-495. https://doi.org/10.1038/990031
  23. Igarashi, Y., Y. Yoshiba, Y. Sanada, K. Yamaguchi Shinozaki, K. Wada and K. Shinozaki. 1997. Characterization of the gene for $\Delta^{1}$-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa. Plant Mol. Biol. 33: 857-865. https://doi.org/10.1023/A:1005702408601
  24. Makela, P., J. Kärkkäinen and S. Somersalo. 2000. Effect of glycinebetaine on chloroplast ultrastructure, chlorophyll and protein content, and RuBPCO activities in tomato grown under drought or salinity. Biol. Plant. 43: 471-475. https://doi.org/10.1023/A:1026712426180
  25. Savoure, A., X.J. Hua, N. Bertauche, M. Van Montagu and N. Verbruggen. 1997. Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana. Mol. Gen. Genet. 254: 104-109. https://doi.org/10.1007/s004380050397
  26. Uberbacher, E.C. and R.J. Mural. 1991. Locating proteincoding regions in human DNA sequences by a multiple sensor-neural network approach. Proc. Nat. Acad. Sci. USA. 88: 11261-11265. https://doi.org/10.1073/pnas.88.24.11261
  27. Zimmermann, U. 1978. Physics of turgor- and osmoregulation. Annu. Rev. Plant Physiol. 29: 121-148. https://doi.org/10.1146/annurev.pp.29.060178.001005