Heterologous Expression of Arabidopsis Purple Acid Phosphatase (AtPAP15) in Tobacco for Remediation of Excess Phosphorus from Soil.

Heterologous expression of Arabidopsis purple acid phosphatase (AtPAP15) in tobacco for remediation of excess phosphorus from soil

Jane Bartonjo, SinilalBhaskaran, DeveshShukla,Sneha Murthy and ShivendraSahi, Department of Biology, Western Kentucky University, Bowling Green, KY

Abstract

Phosphorus is a key macronutrient element that plays vital role in growth and development of the plants. Extensive exposure of livestock manure, phosphorus fertilizers, industrial waste as well as leaching of phosphorus from mining sites results in accumulation of phosphorus in soil. Escape of this excess phosphorus into nearby water bodies with runaway water creates environmental issues like eutrophication. Phytoremediation is considered an efficient, cost effective and environmentally friendly technology for removal of pollutants from soil. Use of transgenic plants overexpressing phosphorus-metabolizing genes like phytase genes may enhance the uptake and utilization of soil phosphorus. In order to test the feasibility of this strategy, purple acid phosphatase gene from Arabidopsis thaliana (AtPAP15) was isolated and cloned to overexpress in Nicotiana tabacum. Full length cDNA encoding AtPAP15 was amplified and cloned under the control of CaMV35S promoter in plant transformation vector pBIN19. Transformation of N. tabacum was performed using Agrobacterium tumefaciens strain EHA105 carrying the recombinant plasmid. Plants regenerated in selection medium were screened through PCR for the presence of AtPAP15using gene specific primers. PCR positive plants (F₀ generation) were transferred to greenhouse for production of F₁ generation plants. The expression level of AtPAP15 gene in different transgenic lines will be quantified using Real Time PCR to select the overexpressing lines. F₁ plants will be further screened for AtPAP15 insert and/or copy number using southern blot. Alternatively, Southern hybridization will be carried out to determine the copy number of the inserted AtPAP15 gene.This will be followed by studies on organic phosphorous uptake using inductively coupled plasma optical emission spectrometry.