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RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

Peng T, Jia MM, Liu JH - Front Plant Sci (2015)

Bottom Line: PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf.When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content.In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University, Ganzhou China.

ABSTRACT
Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

No MeSH data available.


Related in: MedlinePlus

Transformation, regeneration, and characterization of trifoliate orange transgenic plants. (A,B) Culture of the stem segments on selection medium for 30 d (A) and 60 d (B), respectively. (C) Regeneration of kanamycin -resistant shoots on the selection medium. (D) A rooting plant on the root-inducing medium. (E) Genomic PCR of the kanamycin-resistant plants using designed primers specific to PtrPRP (upper) and NPTII (bottom), respectively. (F) Expression analysis of PtrPRP in six positive transgenic plants, as revealed by RT-PCR. Actin gene was used as an internal control. (G) Analysis of PtrPRP expression level in RNAi-51 using qRT-PCR.
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Figure 6: Transformation, regeneration, and characterization of trifoliate orange transgenic plants. (A,B) Culture of the stem segments on selection medium for 30 d (A) and 60 d (B), respectively. (C) Regeneration of kanamycin -resistant shoots on the selection medium. (D) A rooting plant on the root-inducing medium. (E) Genomic PCR of the kanamycin-resistant plants using designed primers specific to PtrPRP (upper) and NPTII (bottom), respectively. (F) Expression analysis of PtrPRP in six positive transgenic plants, as revealed by RT-PCR. Actin gene was used as an internal control. (G) Analysis of PtrPRP expression level in RNAi-51 using qRT-PCR.

Mentions: To elucidate the role of PtrPRP in cold tolerance, RNAi strategy was used to suppress PtrPRP in trifoliate orange. For this purpose, a 253-bp cDNA fragment displaying lower degree of sequence conservation among C-terminal ends of the PRPs was used to construct PtrPRP-RNAi vector, which was then transferred into trifoliate orange via Agrobacterium-mediated transformation (Figures 6A–D). Kanamycin-resistant plants were confirmed to be positive using genomic PCR (Figure 6E). Semi-quantitative RT-PCR assay indicated that among the positive lines PtrPRP was successfully down-regulated in three lines, and the greatest suppression was observed in lines #51 and #52, which were hereafter designated as RNAi-51 and RNAi-52, respectively (Figure 6F). Expression of PtrPRP in RNAi-51 was also checked using qRT-PCR, and the results confirmed that PtrPRP is trully knocked down, purporting the semi-quantitative RT-PCR data (Figure 6G). The two RNAi lines, RNAi-51 and RNAi-52, showed no difference in plant morphology in comparison with the WT.


RNAi-based functional elucidation of PtrPRP, a gene encoding a hybrid proline rich protein, in cold tolerance of Poncirus trifoliata.

Peng T, Jia MM, Liu JH - Front Plant Sci (2015)

Transformation, regeneration, and characterization of trifoliate orange transgenic plants. (A,B) Culture of the stem segments on selection medium for 30 d (A) and 60 d (B), respectively. (C) Regeneration of kanamycin -resistant shoots on the selection medium. (D) A rooting plant on the root-inducing medium. (E) Genomic PCR of the kanamycin-resistant plants using designed primers specific to PtrPRP (upper) and NPTII (bottom), respectively. (F) Expression analysis of PtrPRP in six positive transgenic plants, as revealed by RT-PCR. Actin gene was used as an internal control. (G) Analysis of PtrPRP expression level in RNAi-51 using qRT-PCR.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587090&req=5

Figure 6: Transformation, regeneration, and characterization of trifoliate orange transgenic plants. (A,B) Culture of the stem segments on selection medium for 30 d (A) and 60 d (B), respectively. (C) Regeneration of kanamycin -resistant shoots on the selection medium. (D) A rooting plant on the root-inducing medium. (E) Genomic PCR of the kanamycin-resistant plants using designed primers specific to PtrPRP (upper) and NPTII (bottom), respectively. (F) Expression analysis of PtrPRP in six positive transgenic plants, as revealed by RT-PCR. Actin gene was used as an internal control. (G) Analysis of PtrPRP expression level in RNAi-51 using qRT-PCR.
Mentions: To elucidate the role of PtrPRP in cold tolerance, RNAi strategy was used to suppress PtrPRP in trifoliate orange. For this purpose, a 253-bp cDNA fragment displaying lower degree of sequence conservation among C-terminal ends of the PRPs was used to construct PtrPRP-RNAi vector, which was then transferred into trifoliate orange via Agrobacterium-mediated transformation (Figures 6A–D). Kanamycin-resistant plants were confirmed to be positive using genomic PCR (Figure 6E). Semi-quantitative RT-PCR assay indicated that among the positive lines PtrPRP was successfully down-regulated in three lines, and the greatest suppression was observed in lines #51 and #52, which were hereafter designated as RNAi-51 and RNAi-52, respectively (Figure 6F). Expression of PtrPRP in RNAi-51 was also checked using qRT-PCR, and the results confirmed that PtrPRP is trully knocked down, purporting the semi-quantitative RT-PCR data (Figure 6G). The two RNAi lines, RNAi-51 and RNAi-52, showed no difference in plant morphology in comparison with the WT.

Bottom Line: PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf.When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content.In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University, Ganzhou China.

ABSTRACT
Hybrid proline-rich proteins (HyPRPs) have been suggested to play important roles in various plant development and stress response. In this study, we report the cloning and functional analysis of PtrPRP, a HyPRP-encoding gene of Poncirus trifoliata. PtrPRP contains 176 amino acids, among which 21% are proline residues, and has an 8-cysteine motif (8 CM) domain at the C terminal, a signal peptide and a proline-rich region at the N terminal. PtrPRP is constitutively expressed in root, stem and leaf, with the highest expression levels in leaf. It was progressively induced by cold, but transiently upregulated by salt and ABA. Transgenic P. trifoliata plants with knock-down PtrPRP by RNA interference (RNAi) were generated to investigate the role of PtrPRP in cold tolerance. When challenged by low temperature, the PtrPRP-RNAi plants displayed more sensitive performance compared with wild type (WT), as shown by higher electrolyte leakage and malondialdehyde content. In addition, the RNAi lines accumulated more reactive oxygen species (ROS) and lower levels of proline relative to WT. These results suggested that PtrPRP might be positively involved in cold tolerance by maintaining membrane integrity and ROS homeostasis.

No MeSH data available.


Related in: MedlinePlus