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Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii - PubMed

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii

Zhangli Hu et al. PLoS One. 2012.

Abstract

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Schematic diagram of homologous recombination events between pBsLPNCB and CC-2654 mtDNA.

The dotted line represents the deletion part of mtDNA in CC-2654 relative to wild-type CC-124. The empty boxes are the mitochondrial genes and the empty arrows above the genes indicate their transcription directions. The crossed solid lines denote the homologous recombination region between the CC-2654 mtDNA and expression vector pBsLPNCB.

Figure 2
Figure 2. Growth of transformants MT-Bs on the TAP media with 3 µg/mL Zeomycin (A) and PCR analysis with B1/B2 primers (B).

M, DL2000 marker; lanes1-6, different clones of MT-B; lanes 7–8, negative controls CC-2654 and CC-124; lane 9, water used as negative control.

Figure 3
Figure 3. Analysis of the Sh ble gene integrated site in the mitochondrial genome of MT-B.

A: The Southern hybridization of C. reinhardtii MT-B with the ble-probe; B: Restriction map of the mitochondrial genome of C. reinhardtii MT-B showing the positions of all mapped genes and restriction sites. Line 1,2, 3: Total DNA of MT-B were digested with Bam HI, Nde I and Sac II, respectively, Line 4: the genome of CC-2654 digested with NdeI was used as control.

Figure 4
Figure 4. Sh ble gene transcripts in transformant MT-B.

Panal A is the RT-PCR analysis of MT-B strains and PCR amplification with total RNA. M: DL2000 marker, Lane 1: cDNA of negative control CC-2654 used as template; 2–4: total RNA isolated from different MT-B strains used as template; lane 5–7: cDNA of different MT-B strains used as template. Panal B is the Northern blot analysis of C. reinhardtii MT-B. Lane 1, total RNA of MT-B was detected with Sh ble probe; lane 2. total RNA of CC-2654 was used as negative control.

Figure 5
Figure 5. Western blotting analysis of the transformants MT-B using monoclonal anti-BLE antibody.

The soluble protein of CC-2654 (2) were used as control, the soluble protein of transformants MT-B (1) was detected by Western blotting.

Figure 6
Figure 6. Sensitivity analysis of transgenic strains MT-Bs to Zeomycin.

a: wild-type strain CC-124; b: respiratory deficient strain CC-2654; c: transgenic strain MT-B; d: empty control.

Figure 7
Figure 7. The expression levels of the Sh ble gene and the mitochondrial genes in transgenic MT-B.

A: The gene organization of MT-B mitochondrial genome: B: RT-PCR results of Sh ble gene and the mitochondrial genes with rrnL7 as internal control; C: Relative mRNA levels analysis using BIO-RAD image software.

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