Q.60 Which of the following statements are TRUE on transgene approach?
P. T–DNA integration occurs mainly through non–homologous recombination
Q. The Gateway cloning depends on recombination technology as opposed to standard uses of
restriction enzymes and DNA ligase
R. The localization of β–glucuronidase (GUS) activity as a result of expression of GUS reporter
gene can be visualized in a histochemical assay using the X–gal
S. The green fluorescent protein gene (GFP) is isolated from the bacterium Photinus pyralis
(A) P, Q (B) Q, R (C) P, S (D) R, S
P and Q are true statements on transgene approaches.
Question Breakdown
This GATE Life Sciences question tests molecular biology techniques in plant transgenics. Transgenes integrate via Agrobacterium T-DNA or direct methods, using reporter genes (GUS/GFP) and cloning systems like Gateway for efficient construct assembly.
Statement Analysis
P: T-DNA integration occurs mainly through non-homologous recombination
True. Agrobacterium T-DNA integrates randomly via host NHEJ pathway (Ku70/80, Lig4), creating small deletions/fillers at junctions, not precise HR.
Q: Gateway cloning depends on recombination, not restriction enzymes/ligase
True. Gateway uses λ phage att site-specific recombination (BP/LR Clonase) for directional cloning between Entry/Destination vectors, avoiding RE digestion.
R: GUS activity visualized histochemically using X-gal
False. GUS (β-glucuronidase) uses X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) for blue precipitate; X-gal is for β-galactosidase (lacZ reporter).
S: GFP gene isolated from Photinus pyralis
False. GFP from jellyfish Aequorea victoria (aequorin); luciferase (firefly light) from Photinus pyralis.
Correct Choice
(A) P, Q
Transgene approach true statements clarify T-DNA integration mechanisms and Gateway cloning for GATE Life Sciences plant biotech preparation.
Key Transgene Technologies
-
T-DNA: NHEJ-mediated random insertion (90%+ cases)
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Gateway: attB×attP → attL → attR recombination cassette
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GUS reporter: X-Gluc substrate, blue histochemical stain
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GFP: Aequorea victoria, green fluorescence (488 nm excitation)
Truth Table
| Statement | Content | True? | Reason |
|---|---|---|---|
| P | T-DNA non-homologous recombination | Yes | NHEJ pathway dominant |
| Q | Gateway recombination-based | Yes | No RE/ligase needed |
| R | GUS with X-gal | No | X-Gluc for GUS; X-gal for lacZ |
| S | GFP from Photinus pyralis | No | Jellyfish source |
GATE Exam Relevance
XL-Molecular Biology PYQ tests protocol specifics. Memorize: T-DNA NHEJ (not HR), Gateway (Invitrogen), GUS-X-Gluc, GFP-jellyfish, luciferase-firefly. Common in transformation questions.
