Ptcorp gene induced by cold stress was identified by proteomic analysis in leaves of Poncirus trifoliata (L.) Raf.
- 263 Downloads
A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.
KeywordsPoncirus trifoliata Mass spectrometry Cold stress Resistance protein 2-D IEF/SDS-PAGE Ptcorp
The research was supported by the National High Technology Research and Development Program of China (863 Program, no. 2011AA100205) and the project 09WD48 funded by Hunan Agricultural University, Hunan, China.
- 1.Kender JW (2003) Citrus. HortScience 38:1043–1047Google Scholar
- 2.Shen DX, Wang YY, Chen LG (1997) Genetics and breeding in citrus. Science Press, Beijing, p 289Google Scholar
- 3.Cao Q, Kong WF, Wen PF (2004) Plant freezing tolerance and genes express in cold acclimation. Acta Ecol Sin 24:806–811Google Scholar
- 6.Long GY, Liu J, Rao LQ, Deng ZN, Xiong XY, Jiao L (2006) Application of mRNA differentially display technique in gene expression in plants. Sci Res Mon 21:25–27Google Scholar
- 11.Sanchez-Ballesta MT, Rodrigo MJ, Lafuente MT, Granell A, Zacarias L (2004) Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the Flavedo of fruit but responsive to cold and water stress in leaves. J Agric Food Chem 52:1950–1957PubMedCrossRefGoogle Scholar
- 18.Hajduch M, Rakwal R, Agrawal GK, Yonekura M, Pretova A (2001) High-resolution two-dimentional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentations of ribulose-1, 5-bisphosphate carbosylate/oxygenase and induction of stress-related proteins. Electrophoresis 22:2824–2831PubMedCrossRefGoogle Scholar
- 23.Long GY, Liu J, Rao LQ (2005) Methods of plant proteome research. J Hunan Agric Univ 31:342–346Google Scholar
- 26.Yelenosky G (1985) Cold hardiness in citrus. Hortic Rev 7:201–238Google Scholar
- 27.Liu J, Long GY, Rao LQ, Fan S, Yang H, Peng GP, Jiang FJ, Li LL (2006) Study on the lethal temperature of Poncirus trifoliata Raf. and Citrus reticalata Blanco cv. Miyamoto seedlings with different cold-resistance during growth period. Life Sci Res 10:82–86Google Scholar
- 28.Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672PubMedCrossRefGoogle Scholar
- 29.He XH, Li YR, Guo YZ, Tang ZP, Li RB (2005) Genetic analysis of 23 mango cultivars collection in Guanxi province revealed by ISSR. Mol Plant Breed 3:829–834Google Scholar
- 32.Liu ZQ, Lin DB (1993) The relationship between specific proteins regulated by ABA/GAs and cold resistance of citrus plants. Acta Hortic Sin 20:335–340Google Scholar
- 33.Lin DB, Liu ZQ (1994) Effect of cold acclimation and ABA on membrane stability and synthesis of membrane protein in citrus. J Nanjing Agric Univ 17:1–5Google Scholar
- 41.van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, Muskens M, Wouters D, Pereira A, Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J 36:867–882PubMedCrossRefGoogle Scholar