Detection and copy number estimation of the transgenic nucleotide sequences in an unknown GM event of Oryza sativa

• Autorzy: Sajjad A.M. , Bashir T. , Saeed S. , Ahmad E.

Warianty tytułu

PL

Detekcja i oszacowanie liczby kopii transgenu cry1Ac kodującego cechę odporności na szkodniki w genetycznie modyfikowanym ryżu (Oryza sativa)

• Języki publikacji: EN

Abstrakty

EN

The present study was designed to establish a qualitative detection method based on conventional and real time PCR assay to screen the commonly grown rice varieties for the presence of the cry1Ac gene. The detection of genetically modified rice in the screening process would necessitate accurate assay development and precise qualitative PCR tests complying with established procedures for the detection and characterization of transgenes in food grains. Such assay would not only enable the monitoring of transgene flow in local agricultural environment but also the characterization of different plant species produced with this transgene and its regulatory components. Thus, a reliable and quick screening assay was established for the qualitative detection of the transgene along with the promoter and selectable marker gene in genetically modified rice. By conventional PCR, a fragment of 215 bp was amplified with gene specific primers of cry1Ac. Primers for other transgenes such as gna and bar were also employed; however, no amplification was detected. The presence of the p35s, sps, and nptII genes was confirmed by qualitative real-time PCR. The specificity of the respective PCR products was checked through melt peak curve analysis. Sharp and precise melting temperatures indicated the presence of a single kind of PCR product in correspondence to each of the primers used. Moreover, the copy number of cry1Ac was estimated by ΔΔCT method. It is proposed that the primer sets and experimental conditions used in this study will be sufficient to meet the requirements for molecular detection and characterization of the cry1Ac transgene and affiliated sequences in sorting out conventional rice varieties from the ones which are genetically modified. It will also help to monitor the ecological flow of these transgenes and other biosafety factors.

PL

Celem niniejszych badań było opracowanie metody detekcji genu cry1Ac, odpowiedzialnego za odporność na szkodniki, z wykorzystaniem konwencjonalnej techniki PCR oraz PCR w czasie rzeczywistym, w celu badania obecności tego transgenu w powszechnie uprawianych odmianach ryżu. Detekcja modyfikacji genetycznej obecnej w odmianach ryżu wymaga opracowania metodyki badawczej z wykorzystaniem metody PCR, zgodnie z ustalonymi procedurami dotyczącymi obecności transgenów w zbożach konsumpcyjnych. Badania te nie tylko umożliwiłyby monitorowanie przepływu transgenów w lokalnym środowisku rolnym, ale również dokonanie charakterystyki różnych gatunków roślin wytworzonych z wykorzystaniem oznaczanego transgenu oraz związanych z nim fragmentów regulatorowych. W niniejszej pracy przedstawiono metodę wiarygodnego testu kontrolnego, w celu detekcji transgenu wraz z promotorem i selekcyjnym genem markerowym. Przy użyciu konwencjonalnej techniki PCR z zastosowaniem starterów specyficznych dla genu cry1Ac powielono fragment długości 215 pz. Poszukiwano także, lecz nie wykryto, innych transgenów, takich jak gna i bar. Obecność genów p35s, sps i nptII została potwierdzona na podstawie techniki ilościowego PCR w czasie rzeczywistym. Poszczególne produkty PCR poddano analizie z wykorzystaniem krzywych topnienia. Ostre wierzchołki krzywych topnienia wskazywały na obecność pojedynczego produktu PCR, odpowiednio dla każdego użytego startera. Za pomocą metody ΔΔCT oszacowano liczbę kopii genu cry1Ac. Na podstawie niniejszych badań wykazano, że zestawy starterów i opracowana metodyka są właściwe do molekularnej detekcji i charakterystyki transgenu cry1Ac oraz sekwencji z nim związanych i mogą służyć do wskazania genetycznie modyfikowanych odmian ryżu. Metoda ta może być również pomocna w monitorowaniu środowiska pod względem przepływu tego transgenu.

• Wydawca: -
• Czasopismo: Acta Agrobotanica
• Rocznik: 2016
• Tom: 69
• Numer: 4
• Opis fizyczny: Article 1684 [14p.], fig.,ref.

Twórcy

autor

Sajjad A.M.

• Institute of Molecular Biology and Biotechnology, The University of Lahore, 1-KM Defence Road, 54000 Lahore, Pakistan

autor

Bashir T.

• Institute of Molecular Biology and Biotechnology, The University of Lahore, 1-KM Defence Road, 54000 Lahore, Pakistan

autor

Saeed S.

• Institute of Molecular Biology and Biotechnology, The University of Lahore, 1-KM Defence Road, 54000 Lahore, Pakistan

autor

Ahmad E.

• Institute of Molecular Biology and Biotechnology, The University of Lahore, 1-KM Defence Road, 54000 Lahore, Pakistan

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Identyfikatory

DOI

10.5586/aa.1684