Unknown,Transcriptomics,Genomics,Proteomics

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Profiling gene expression levels to investigate the degree of substantial equivalence between GM and non-GM potato

ABSTRACT: The goal of this study was to calculate the degree of substantial equivalence between a GM potato line modified for late blight resistance and corresponding non-GM controls in the presence/absence of P. infestans inoculation. The GM phenotype was conferred through the introduction of the Rb gene which has been previously cloned from Solanum bulbocastanum (Song et al. 2003). The resulting GM phenotype was characterised by the expression of strong, partial resistance to P. infestans. By obtaining an expression profile from a GM and non-GM population before and after exposure to P. infestans, this effectively provides for the completion of two risk assessments: impact on the potato transcriptome of plus/minus genetic modification; before and after exposure to the pathogen. For each biological replicate, individuals from three separate GM lines (A, B, C) and three separate non-GM lines (D, E, F) were randomly sorted and cultivated for analysis. Each GM line contained an independent, single transgene insertion, which was verified through Southern Blot analysis. One day prior to pathogen exposure, the 4-week-old potato plants were transferred into a growth chamber with a 16-h light period, 90% relative humidity at a temperature of 15C. Plants were randomly selected and 14 individuals/line were inoculated with a field isolate of P. infestans. An additional 14 plants/line were sprayed with water. Inoculations were made with a sporangial suspension of 20,000/mL and plants were sprayed until run-off. Twenty-four hours post-inoculation, leaf tissue was collected from 9 plants per treatment, flash frozen in liquid nitrogen and pooled. Total leaf RNA was extracted using Trizol and the whole experiment was repeated three times (three biological replicates). Keywords: Direct comparison 19 hybs total

ORGANISM(S): Solanum tuberosum

SUBMITTER: Jia Liu

PROVIDER: E-GEOD-8258 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objectives: Our work focuses on the responses of Solanaceous plants to viruses that cause economically important diseases in tree fruits. Using mock inoculated leaf tissue as a reference, we plan to compare the gene expression profiles of Nicotiana Benthamiana plants infected with one of three viruses; Plum Pox Potyvirus (PPV), Tomato Ringspot Nepovirus (ToRSV), and Prunus Nectrotic Ringspot Nepovirus (PNRSV). Our goals are as follows: (1) Identify genes that are induced/repressed in response to individual viruses. (2) Identify genes that are induced/repressed in response to all 3 viruses. (3) Compare results to existing potato array data to look for similarities in responses to other pathogens. Experimental Design: Nicotiana benthamiana plants were inoculated with one of three viruses: PPV, ToRSV, or PNRSV. 3 week old plants were inoculated by rubbing virus infected plant sap onto leaves dusted with carborundum. Control plants were mock inoculated using sap from healthy plants. All plants were maintained in a growth chamber at 22C for 18 days. 8 plants were inoculated with each virus or mock inoculated. This experiment was repeated twice. 4 biological replicates derived from 2 virus infected plants from each replica experiment (4 plants) are to be used for hybridizations. RNA from all mock inoculated plants was similarly pooled to create 4 biological replicates. Each replicate control will serve as a universal reference sample that is to be hybridized pair wise with each of the three virus infected samples. RNA extraction: After 18 days, un-inoculated leaves displaying clear symptoms were harvested and immediately frozen in liquid N2. Total RNA was purified using Trizol according to TIGRs listed protocol. RNA was subsequently treated with Turbo DNA-free RNase (Ambion cat#1907). Finally, total RNA was further purified on RNeasy columns (Qiagen) according to manufacturer’s instructions and quantified using a Nanodrop spectrophotometer. Keywords: Reference design 23 hybs total

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Profiling gene expression levels to investigate the degree of substantial equivalence between GM and non-GM potato

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