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, 8 (3), 140

Construction of Chloroplast Transformation Vector and Its Functional Evaluation in Momordica charantia L

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Construction of Chloroplast Transformation Vector and Its Functional Evaluation in Momordica charantia L

Muralikrishna Narra et al. 3 Biotech.

Abstract

Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3'-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.

Keywords: Chloroplast transformation vector; Expression cassette; Flanking regions; Plastome sequence.

Conflict of interest statement

Compliance with ethical standardsThe authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic representation of plastid vector construction. a Physical map of inverted repeat region of C.sativus chloroplast genome used for vector construction. b Flanking sequences trnI/trnA genes with respective restriction sites at their cut ends inserted into pKS+II. c aadA expression cassette having promoter PsbA and terminator PsbA. d Chloroplast transformation vector carrying a selectable marker with two flanking regions
Fig. 2
Fig. 2
CuIA plasmid assembly and confirmation of vector. a Amplified products of trnA (lane: 1) and trnI (lane: 2) flanking sequences with 1.570 and 1.530 kb sizes, respectively. b Amplified product of aadA (lane: 2) with 1.1 kb size. c Double restriction digestion of trnI flank (pLI1), Lane: 1 Undigested pKS + plasmid, Lane: 2 Restriction digestion of pKS + plasmid with SacI resulting in 3.0 kb fragment, Lanes: 3 & 4 Double restriction digestion of trnI flank with SacI and SacII resulted in 3.0 & 1.5 kb fragments. d Colony PCR confirming the ligation of trnI, resulted in 1.5 kb fragment with M13 primers. e Restriction digestion of pLIA2 plasmid having trnI and trnA flanks, Lane: 1 Undigested pKS+backbone vector, Lane: 2 pKS + digested with SacI resulting in 3.0 kb size fragment, Lane: 3 Plasmid pLI1 digested with SacI resulting in 4.2 kb size fragment, Lane: 4 Plasmid pLIA2 digested with SalI resulting in 6.0 kb size fragment, Lane: 5 pLI1 digested with SacI & SacII (trnI) resulting in 3.0 & 1.5 kb size fragments, Lane: 6 pLIA2 digested with SalI & KpnI (trnA) resulting in 4.2 & 1.5 kb size fragments, Lane: 7 pLIA2 digested with SacI & SacII (trnI) resulting in 4.2 & 1.5 kb size fragments. f Colony PCR confirming the ligation of trnI & trnA, resulted in 3.0 kb fragment. g Restriction digestion of CuIA plasmid having aadA flanked by trnI and trnA sequences, Lane: 1 pKS+ digested with SacI resulting in 3.0 kb size fragment, Lanes: 2 & 4 Single digestion of CuIA plasmid vector with SacI resulted in 7.1 kb size fragment, Lane: 3 CuIA plasmid digested with SacII and KpnI resulted in 3.0 & 4.2 kb size fragments. h Colony PCR confirming the ligation of aadA along with trnI and trnA flanks, resulted in 4.2 kb fragment
Fig. 3
Fig. 3
Map of CuIA plastid transformation vector and sequence analysis of flanking genes. a Physical map of species-specific vector showing position of different restriction sites. b blastn alignment results of cloned rrn16-trnI flanking gene into the vector showing maximum similarity with genome of Cucumis sativus L. c blastn alignment results of cloned trnA-rrn23 flanking gene into the vector showing maximum similarity with genome of Cucumis sativus L
Fig. 4
Fig. 4
Generation of transplastomic plants. a Petiole segments prior to bombardment. b After 2 days of bombardment, petiole explants were transferred to MS regeneration medium supplemented with 300 mg/L spectinomycin. c Regeneration of spectinomycin-resistant shoot from petiole segment after 45 days of culture on same selection medium. d Elongation of transplastomic shoots on MS regeneration medium containing 300 mg/L spectinomycin
Fig. 5
Fig. 5
Vector map and position of primers used for PCR amplification
Fig. 6
Fig. 6
PCR analysis of plastid transformants of Momardica charantia L. a Confirmation of aadA resulted in 1.1 kb size amplicon fragment. b Confirmation of plastome integration resulting in 3.0 kb in wild and 4.2 kb fragment in transformed lines

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