DOI: 10.1007/s10535-016-0592-8 BIOLOGIA PLANTARUM 60 (2): 285-291, 2016 285 Isolation and characterization of genes encoding lipid transfer proteins in Linum usitatissimum V.A. MHASKE 1 , R. DATLA 2 , S. QIU 2 , and A.M. HARSULKAR 1 * Cell and Molecular Biology Laboratory, Interactive Research School For Health Affairs, Bharati Vidyapeeth University, Pune, India 1 Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, S7N OW9, Canada 2 Abstract Very little is known about lipid transfer proteins from flax (Linum usitatissimum L.). In the present work, three genes encoding a lipid transfer protein (LTP) were isolated from flax, two of which encoded Type-1 and one Type-2 LTPs with molecular masses of about 9 and 7 kDa, respectively. The analysis of deduced amino acid sequence reveals that only Type 2 of the L. usitatissimum leaf specific LTP (LuLTP_Ls) had an N terminal signal peptide consisting of 23 amino acids. The phylogenetic analyses of LuLTP_Ls suggest their closest relatedness with respective proteins from Dimocarpus longan and Vitis vinifera. The gene expression analysis shows that LTP Type 1 genes, which include LuLTP_Ls1 and LuLTP_Ls3, were progressively expressed during leaf development, whereas LuLTP_Ls4 (Type 2) was expressed only at initial and terminal senescence stages of cotyledons. The results suggest that both types of LuLTP_Ls were differentially yet significantly expressed in cotyledons implicating their function in transport and scavenging lipidic skeletons for the benefit of other developing parts of the plant. Additional key words: flax, gene expression, molecular cloning, plant development, RT-qPCR. Introduction Leaf senescence is terminal phase leading to cellular disassembly and mobilization of released materials to newly developing organs. Leaf senescence is not only associated with a passive degradation of macromolecules but also involves active recycling nutrients to support other developing organs (Quirino et al. 2000, Hortensteiner and Feller 2002). Senescence is terminal phase of developmental process that includes highly coordinated changes in gene expression and protein synthesis (Shahri and Tahir 2011). Additionally, underlying signalling and transcriptional networks are up- regulated with initiation of senescence. These signalling programs trigger remobilization of macromolecule breakdown products to sink locations. This macro- molecular transport is mediated via specific transporters, and lipid transfer proteins (LTPs) are involved in signalling and transport of lipidic macromolecules (Edqvist and Farbos 2002). The LTPs are small basic proteins widely distributed in the plant kingdom and are encoded by a multigene family. Non-specific LTPs (ns-LTPs) are ubiquitous and have been reported in various plant species such as rice, peach, cumin, and coffee (Liu et al. 2002, Pasquato et al. 2006, Zaman and Abbasi 2009, Zottich et al. 2011). The Ns-LTPs are classified into two subfamilies as ns- LTP1 (9 kDa) and ns-LTP2 (7 kDa) according to their molecular masses and eight conserved cysteine residues (Kader 1996, Liu et al. 2002). Structurally, LTPs are composed of helices and a long flexible loop at the C terminal end (Lee et al. 1998, Han et al. 2001). The four α-helices enclose a hydrophobic cavity which can specifically accommodate binding lipids and other hydrophobic ligands (Kader 1996, Lascombe et al. 2008). The Ns-LTPs lack a tryptophan residue and consist of conserved C-Xn-C-Xn-CC-Xn-CXC-Xn-C-C cysteine motifs. Plant nsLTPs show different binding affinities for monoacylated as well as diacylated hydrophobic molecules (Han et al. 2001, Charvolin et al. 1999).  Submitted 3 July 2014, last revision 5 October 2015, accepted 14 October 2015. Abbreviations: EST - expressed sequence tag; GAL 1 - galactose inducible 1; His - histidine; LuLTP_Ls - Linum usitatissimum lipid transfer protein; ORF - open reading frame; RT-PCR - reverse transacriptase - polymerase chain reaction. Acknowledgments: Financial assistance to the Indian side by the Department of Biotechnology, Govt. of India and Genome Canada and Genome Prairie funding through the TUFGEN project to Raju Datla is acknowledged. * Corresponding author; e-mail: aharsulkar@yahoo.com