Q.57 You are asked to design a genetic construct for high-level expression of a gene encoding the
therapeutic protein 18 (TP18) via plastid transformation. Select the CORRECT set of genetic elements for
this construct.
(A) Actin1 promoter → TP18 coding sequence → Actin1 transcription terminator
(B) Ubiquitin1 promoter → TP18 coding sequence → Ubiquitin1 transcription terminator
(C) rbcS promoter → TP18 coding sequence → rbcS transcription terminator
(D) rbcL promoter → TP18 coding sequence → rbcL transcription terminator
Plastid transformation enables high-level expression of therapeutic proteins like TP18 by leveraging chloroplast genome features. The correct construct uses plastid-native elements for optimal transcription and stability.
Correct Answer
Option (D) rbcL promoter → TP18 coding sequence → rbcL transcription terminator is correct. The rbcL gene encodes the large subunit of Rubisco, highly expressed in plastids, making its promoter and terminator ideal for driving strong, stable expression of foreign genes like TP18 in chloroplasts.
Why Plastid Promoters Excel
Plastid genomes rely on prokaryotic-like promoters such as Prrn (rRNA operon) or PrbcL, which ensure polycistronic transcription and high copy number (up to 10,000 per cell). Nuclear promoters fail here due to lacking plastid sigma factors and RNA polymerase specificity. Constructs with rbcL elements achieve 10-100 fold higher yields than nuclear ones for therapeutic proteins.
Option Analysis
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(A) Actin1 promoter → TP18 → Actin1 terminator: Actin1 is a nuclear constitutive promoter active in monocots like rice, unsuitable for plastids as it requires nuclear transcription machinery.
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(B) Ubiquitin1 promoter → TP18 → Ubiquitin1 terminator: Ubiquitin1 (Ubi-1) drives high nuclear expression in cereals but does not function in plastids lacking eukaryotic introns and enhancers.
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(C) rbcS promoter → TP18 → rbcS terminator: rbcS encodes Rubisco small subunit, nuclear-encoded with a transit peptide for plastid import; its promoter works in nucleus, not plastid genome.
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(D) rbcL promoter → TP18 → rbcL terminator: Matches plastid biology; rbcL is plastid-encoded, providing endogenous high-expression control for TP18.
| Option | Promoter Origin | Suitability for Plastid Transformation |
|---|---|---|
| A | Nuclear (Actin1) | No – Lacks plastid compatibility |
| B | Nuclear (Ubi1) | No – Nuclear-specific |
| C | Nuclear (rbcS) | No – Requires transit peptide |
| D | Plastid (rbcL) | Yes – Native high expression |
