377     Isolation of two novel isoforms encoding zinc- and copper-transporting P 1B -ATPase from Gouan (Aeluropus littoralis) Leila Rastgoo 1 , Abbas Alemzadeh *2 , and Alireza Afsharifar 3 1 Biotechnology Center, School of Agriculture, Shiraz University, Shiraz, Iran 2 Crop Production and Plant Breeding Department, School of Agriculture, Shiraz University, Shiraz, Iran 3 Plant Prtection Department, School of Agriculture, Shiraz University, Shiraz, Iran * Corresponding author: alemzadeh@shirazu.ac.ir Abstract Two new isogenes encoding heavy metal ATPase have been partially cloned from Aeluropus littoralis genome. One of them was 849 bp in length and related to an isoform which encodes a copper-transporting P 1B -ATPase (AlHMA1), and the other one was 403 bp in length and related to an isoform encoding a zinc-transporting P 1B -ATPase (AlHMA2). When southern blot hybridization was performed under low-stringency, 6 to 7 hybridization bands appeared, indicating that in this plant heavy metal ATPase is encoded by a multigene family. RT-PCR assay displayed that the AlHMA2 highly expresses in leaf and root and its transcription level is the same in both organs. Phylogenetic tree showed that copper transporters from different plants were located in one group and zinc transporters were located in another group, although all heavy metal P 1B -ATPasees have the same function. It means the different isoforms in single plant that involve in transporting of different heavy metals, may come from different sources. Keywords: gene cloning, tissue expression pattern, Gouan, heavy metals ATPase, metal-transporting. Abbreviations: cDNA - Complementary DNA, NCBI- National Center for Biotechnology Information, IPTG- Isopropyl -D-1- thiogalactopyranoside, DDBJ- DNA Data Bank of Japan, CTAB- Cetyl trimethylammonium bromide. Introduction Heavy metals are a group of elements with specific weight higher than 5 g/cm 3 that exhibit metallic properties. Some of them (Co, Cu, Fe, Mn, Mo, Ni and Zn) known as essential micronutrients because they are vital for plant normal growth as well as they have many biological and structural functions as metalloproteins or metabolites. In contrast, no physiological function has reported for Cd, Cr, Hg and Pb that thought to be toxic and non-essential for plants (Michalak, 2006). Copper as a redox-active transition metal has many functions such as cofactor for many enzymes, in photosynthesis and respiration precesses, signaling of transcription and hormens, protein trafficking machinery, lignin formation in cell walls, and oxidative stress responses (Hirayama et al., 1999; Yruela, 2005). But in high concentration, this element can be toxic for plants in several ways by oxidative damage to biomolecules, decrease in availability and absorption of other essential mineral nutrients and reduce the efficiency of enzymes by binding to enzyme SH groups (Yruela, 2005). Zinc is another essential minor element which has many functional, regulatory and structural roles in many enzymes (Brennan and Bolland, 2006). This element is also important for auxin production, carbohydrate and protein metabolism, protecting cells against oxidative stress, photosynthesis reaction and maintaining the membrane structure and functions (Brennan and Bolland, 2006). Zn toxicity induces chlorosis in plant by competition with the uptake of other elements or reduction in chlorophyll synthesis due to Fe-deficiency (Broadley et al., 2007). So plants developed a number of different hemostasis mechanisms to maintain heavy metals concentrations at cellular level. These mechanisms comprised of regulation of transport, chelation and compartmentalization (Clemens, 2001). Previous studies reported several types of heavy metal transporters that have different sub-cellular location, metal specificity and expression pattern (Colangelo and Guerinot, 2006). The most important role in metal efflux from cytoplasm identified for P 1B -ATPases family. P 1B -ATPases are a subfamily of P-type ATPases that transport heavy metals (Cu + , Cu 2+ , Zn 2+ , Co 2+ , Cd 2+ and Pb 2+ ) against their electrochemical gradient by the energy of ATP hydrolysis (Williams and Mills, 2005). P 1B -ATPases observed in both prokaryotic and eukaryotic species. Plants have more number of P 1B -ATPases in comparison with other organisms (Williams and Mills, 2005). Also plants are the only eukaryotic species which possess Zn 2+ -ATPase. Plant P 1B - ATPase has important roles in nutrition and detoxification (Colangelo and Guerinot, 2006). Previous studies have revealed that most P 1B -ATPases have eight transmembrane domains (TMs), signature CPX motif (CPC, CPH, CPS, SPC and TPC) in sixth domain, with a key role in metals translocation, and carboxyl- or amino-terminal regions (N- MBD and C-MBD) with putative metal-binding domains (Argüello, 2003). Also there are two cytoplasmic loops between helices 4-5 (A domains) and 6-7 (P and N domains). According to substrate specificity and phylogenetic analyses, P 1B -ATPases are grouped into two subgroups: monovalent cation transporter Cu + /Ag + and divalent cation transporter (Zn 2+ /Cd 2+ /Pb 2+ /Co 2+ ) (Axelsen and Palmgren, 2001). Previous studies showed that P 1B -ATPases play a key role in heavy metals detoxification by sending out from cytoplasm.