Homology-based cloning and expression analysis of Rf genes encoding PPR-containing proteins in tobacco

Abstract

ABSTRACT. As a model plant, mechanisms of the cytoplasmic male sterility/restoration of fertility (CMS/Rf) system in tobacco are seldom studied. Using Rf gene sequences from other Solanaceae plants and the draft genome of Nicotiana benthamiana, degenerate primers were designed to amplify the cDNA pool of N. tomentosiformis. In total, six possible Rf sequences were identified, two of which contained basedeletion mutations. The other four were intact open reading frames, of which NtomPPR5 harbored a 3-pentatricopeptide repeat (PPR) motif deletion. Structure analysis revealed that they all encoded a PPRcontaining protein with putative mitochondrial targeting signals at their N-terminus, and they all belong to the P subfamily. Phylogenetic analysis showed that all of the Rf-coding PPRs clustered together, and recent duplication events might have occurred in tobacco after the divergence of the species. Quantitative reverse transcription polymerase Cloning and expression analysis of Rf genes in tobacco chain reaction analysis demonstrated that the NtomRfs were expressed in all tissues of N. tomentosiformis and (CMS) K326, although the expression levels varied with gene, organ, and developmental stage. Furthermore, the expression levels of Rf sequences in K326 were lower than those in CMS K326. The molecular basis of the CMS/Rf system in tobacco requires further investigation.

Expression of a Chromoplast-Specific Lycopene β-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat

“…In this study, a CYC-B gene orthologue was successfully identified from the loquat using a homology-based cloning method, a simple, fast, and efficient method that uses highly conserved sequences to design degenerate primers to clone target genes. This has been used for carrots [34], Phalaenopsis Hybrida [35], and tobacco [36], etc.…”

Interspecific cross‐hybrids of Nicotiana tabacum L. cv. (gla.) S ‘K326’ with Nicotiana alata

“…Fertility restoration genes in N. alata were amplified using one of the degenerate Rf primer couple (Forward: 5′‐ATGACGAGAAYTTCTMTGCTKCGT‐3; Reverse: 5‐TCACT TWTTACTTCCCGAGTGAAA‐3) previously designed by Ding et al. () in N. tomentosiformis . Amplification was performed in 20 μ l volume reaction including 10 μ l of 2× Power Taq PCR Mastermix (BioTeke), 2 μ l of forward and reverse primers (10 μ m each), 2 μ l of cDNA (approximately 50 ng/ μ l) and 6 μ l of deionized water.…”

“…Reverse: 5-TCACT TWTTACTTCCCGAGTGAAA-3) previously designed by Ding et al (2014) in N. tomentosiformis. Amplification was performed in 20 ll volume reaction including 10 ll of 29 Power Taq PCR Mastermix (BioTeke), 2 ll of forward and reverse primers (10 lM each), 2 ll of cDNA (approximately 50 ng/ll) and 6 ll of deionized water.…”

“…Fertility restoration genes in many species have been isolated (Chen and Liu ); however, for Nicotiana, PPR has only been explored in N. benthamiana by Ding et al. (). It has been deduced that dominant fertility restoration gene is only existent in wild Nicotiana species, such as N. repanda and N. debneyi (Burns et al.…”

“…Consequently, it is extremely critical to exploit resources with fertility restoring gene and restore fertility in hybrid (Bentolila et al 2002), and most of the fertility restoration genes encode pentatricopeptide repeat (PPR) containing proteins, which target the mitochondria or plastids for their function (Saha et al 2007). Fertility restoration genes in many species have been isolated ; however, for Nicotiana, PPR has only been explored in N. benthamiana by Ding et al (2014). It has been deduced that dominant fertility restoration gene is only existent in wild Nicotiana species, such as N. repanda and N. debneyi (Burns et al 1978, Reed andBurns 1986).…”