The Lens culinaris Zip gene family : Identification, characterization & gene expression

Abstract

Lentil (Lens culinaris) is an important cool season food legume crop. Lentils provide a rich and inexpensive source of protein, carbohydrates, antioxidant compounds, fibers, minerals, micronutrients (Fe, Zn, Cu, and Mn) macronutrients (P, K, Ca, Mg, and Na), trace elements (Al, Cr, Ni, Pb, Co, Se, Mo) and vitamins are a major source of nutrition in developing nations. Zinc is essential for all forms of life on the planet and plays a significant role in normal growth, development, and reproduction in plants. Lentil is considered to be one of the cheapest sources of dietary Zn, which plays a major role for normal growth, development, and activity of various enzymes in humans and animals. But Zinc deficiency is also widespread, with an estimated 48% of humans at risk, especially populations consuming vegetarian diets rich in unrefined cereals. The Zrt and Irt-like protein (ZIP) family is well characterized for its role in Zn transport and to a lesser extent it role in Fe transport. Thus, for understanding about zinc transport in L.culinaris a detailed knowledge of zip gene is required. In the present study, primers specific to zip gene were designed by IDT Tool and then validated by Primer-BLAST online tool. For providing all the macronutrients and micronutrients to the L.culinaris seedlings, a hydroponic nutrient solution i.e. Hoagland solution is used. Total RNA from seedlings was extracted followed by cDNA synthesis.The zip gene is identified by PCR with amplicon length ~150bp and send for sequencing. Two sequences L100_24_FP2_A05.ab1 (214bp) and L100_24_RP2_B05.ab1 (218bp) were obtained and submitted to NCBI with Accession number. The results were further analysed by in silico analysis i.e. BioEdit and MEGA 5. Nucleotide and amino acid composition were obtained and phlogenetic tree was constructed. The expression analysis was performed by Q-PCR using cox gene as an control. Gene expression was seen by using cDNA from all treated and untreated L.culinaris seedlings when subjected to different concentrations i.e., 100mM, 150Mm and 200 mM for 6hr, 12hr and 24 hrs respectively. The expression of zip gene of zinc treated versus control(untreated) of seedlings was calculated using BioRad IQ-5 software. Expression analysis through Q-PCR (Real time PCR) revealed that the level of zip gene expression was highest at 100 mM for 24 hr (10.06 fold) followed by zinc treatment at 150 mM for 24 hr (8.93 fold) and 200 mM 6 hr (7.06) and lowest at 150 mM for 12hr (3.06 fold). From such type of studies it can be concluded that identifying and characterizing such genes will be helpful in biofortification studies.