Copper (Cu) is a necessary microelement for plants. However, high concentrations of Cu are toxic to plants that change the regulation of several stress-induced proteins. In this study, an annealing control primer (ACP) based approach was used to identify differentially expressed Cu-induced genes in alfalfa leaves. Two-week-old alfalfa plants (Medicago sativa L.) were exposed to Cu for 6 h. Total RNAs were isolated from treated and control leaves followed by ACP-based PCR technique. Using GeneFishing ACPs, we obtained several genes those expression levels were induced by Cu. Finally, we identified several genes including UDP-glucuronic acid decarboxylase, transmembrane protein, small heat shock protein, C-type cytochrome biogenesis protein, mitochondrial 2-oxoglutarate, and trans-2,3-enoyl-CoA reductase in alfalfa leaves. These identified genes have putative functions in cellular processes such as cell wall structural rearrangements, transduction, stress tolerance, heme transport, carbon and nitrogen assimilation, and lipid biosynthesis. Response of Cu-induced genes and their identification in alfalfa would be useful for molecular breeding to improve alfalfa with tolerance to heavy metals.

Ⅰ. INTRODUCTION
 Ⅱ. MATERIALS AND METHODS
  1. Plant material and copper treatment
  2. Isolation of RNA, first strand cDNA synthesis andACP-based GeneFishing
  3. Gene cloning sequencing and bioinformatics analysis
 Ⅲ. RESULTS AND DISCUSSION
 Ⅳ. CONCLUSION
 Ⅴ. ACKNOWLEDGEMENTS
 Ⅵ. REFERENCES

• Ki-Won Lee(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Sang-Hoon Lee(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Ki-Yong Kim(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Hee Chung Ji(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Hyung Soo Park(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Tae Young Hwang(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Gi Jun Choi(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea)
• Md Atikur Rahman(Grassland & Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea) Corresponding author