Abstract
The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. To investigate gene expression in harvested alfalfa, cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11 gene, which has high homology to a putative protein of unknown function in Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a wounding treatment. Northern blot analysis confirmed that the expression patterns of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In addition, genomic walking was performed to isolate the 5′ flanking region of the hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants of M. truncatula and tobacco, GUS gene expression was observed in harvested tissue, especially in the transgenic tobacco plants, but not in the non-harvested control tissue.
Similar content being viewed by others
References
Samac DA, Austin-Phillips S (2006) Alfalfa (Medicago sativa L.). Methods Mol Biol 343:301–311
Barnes DK, Goplen BP, Baylor JE (1988) “Highlights in the USA and Canada” in alfalfa and alfalfa improvement. American Society of Agronomy, Inc., Madison, pp 1–22
Ferullo J, Vezina L, Castonguay Y, Allard G, Madeau P, Willemot C, Laberge S (1996) Post-harvest alteration of in vitro translatable mRNA population in alfalfa (Medicago sativa L.). Crop Sci 36:1011–1016
Blumberg B, Belmonte JC (2008) Subtractive hybridization and construction of cDNA libraries. Methods Mol Biol 461:569–587
Straus D, Ausubel FM (1990) Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc Natl Acad Sci USA 87:1889–1893
Brock GJ, Huang TH, Chen CM, Johnson KJ (2001) A novel technique for the identification of CpG islands exhibiting altered methylation patterns. Nucl Acids Res 29:e123
Parsons YN, Panagea S, Smart CM, Walshaw MJ, Hart A, Winstanley C (2002) Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. J Clin Microbiol 40:4607–4611
Lev S, Horwitz BA (2003) A mitogen-activated protein kinase pathway modulates the expression of two cellulose genes in Cochliobolus heterostrophus during plant infection. Plant Cell 15:835–844
Matvienko M, Torres MJ, Yoder JI (2001) Transcriptional responses in the hemiparasitic plant Triphysaria versicolor to host plant signals. Plant Physiol 127:272–282
Zhang L, Li FG, Liu CL, Zhang CJ, Zhang XY (2009) Construction and analysis of cotton (Gossypium arboreum L.) drought-related cDNA library. BMC Res Notes 2:120
Sahu BB, Shaw BP (2009) Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization. BMC Plant Biol 9:69
Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin H, Chiou TJ, Katagi H, Dewbre GR, Weigel D, Harrison MJ (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541
Sambrook J, Russell DW (2001) Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcription activators in abscisic acid signalling. Plant Cell 15:63–78
Shen X, Martens S, Chen M, Li D, Dong J, Wang T (2010) Cloning and characterization of a functional flavanone-3ss-hydroxylase gene from Medicago truncatula. Mol Biol Rep. doi:10.1007/s11033-009-9913-8
Jin H, Sun Y, Yang Q, Chao Y, Kang J, Jin H, Li Y, Margaret G (2010) Screening of genes induced by salt stress from Alfalfa. Mol Biol Rep 37:745–753
Chao Y, Kang J, Sun Y, Yang Q, Wang P, Wu M, Li Y, Long R, Qin Z (2009) Molecular cloning and characterization of a novel gene encoding zinc finger protein from Medicago sativa L. Mol Biol Rep 36:2315–2321
Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6
Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971
Plewczynski D, Slabinski L, Ginalski K, Rychlewski L (2008) Prediction of signal peptides in protein sequences by neural networks. Acta Biochim Pol 55:261–267
Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10:88–94
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803
von Koskull-Döring P, Scharf KD, Nover L (2007) The diversity of plant heat stress transcription factors. Trends Plant Sci 12:452–457
Schöffl F, Prändel R, Reindel A (1998) Regulation of heat shock response. Plant Physiol 117:1135–1141
Haralampidis K, Milioni D, Rigas S, Hatzopoulos P (2002) Combinatorial interaction of cis elements specifies the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiol 129:1138–1149
Guo L, Chen S, Liu K, Liu Y, Ni L, Zhang K, Zhang L (2008) Isolation of heat shock factor HsfA1a-binding sites in vivo revealed variations of heat shock elements in Arabidopsis thaliana. Plant Cell Physiol 49:1306–1315
Acknowledgements
The authors gratefully acknowledge support for this research from the Canadian Natural Sciences and Engineering Research Council, Strategic Grants Program.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, J., Xiong, AS. & Erickson, L.R. Isolation and characterization of a harvest-inducible gene hi11 and its promoter from alfalfa. Mol Biol Rep 38, 23–29 (2011). https://doi.org/10.1007/s11033-010-0073-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-010-0073-7