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BraLTP1, a lipid transfer protein gene involved in epicuticular wax deposition, cell proliferation and flower development in Brassica napus.

Liu F, Xiong X, Wu L, Fu D, Hayward A, Zeng X, Cao Y, Wu Y, Li Y, Wu G - PLoS ONE (2014)

Bottom Line: Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type.This was consistent with altered expression of a a number of ABC model genes related to flower development.Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.

ABSTRACT
Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

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Basic protein characteristics of BraLTP1 and vector construction.(A) Analysis of the deduced amino acid sequences of BraLTP1 with its homologous sequences in other cruciferae; variable sites (dark grey)the nsLTP-like conserved 8 CM domain (light gray) with conserved cysteine residues (asterisks) and putative extracellular secretory signals (underlined). Sequences are from Arabidopsis thaliana AtLTP1 (AT4g30880), B. rapa BraLTP1 (Bra011229) [32], B. oleracea BolLTP1 (Bol018048) [60], B. napus BnaLTP1 (AY208878), and B. napus BnaLTP2 (KM062522). (B) T-DNA region of the BnaLTP1 overexpression construct containing BnaLTP1 driven by the CaMV 35S promoter. LB = Left border, RB = Right border, poly A = poly A terminator, nptII = kanamycin resistance, NOS Ter = nopaline synthase terminator, NOS Pro = nopaline synthase promoter. (C) Analysis of BraLTP1 mRNA levels in 10-week-old wild type (WT) and T0 35S::BraLTP1 transgenic plants by qRTPCR. Transgenic plants include BraLTP1-1, -6, -7, -19, -20, -21, -22, -28, -29, -32, -34 and -39. Standard errors were derived from three repeated experiment for the expression levels of each plants.
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pone-0110272-g001: Basic protein characteristics of BraLTP1 and vector construction.(A) Analysis of the deduced amino acid sequences of BraLTP1 with its homologous sequences in other cruciferae; variable sites (dark grey)the nsLTP-like conserved 8 CM domain (light gray) with conserved cysteine residues (asterisks) and putative extracellular secretory signals (underlined). Sequences are from Arabidopsis thaliana AtLTP1 (AT4g30880), B. rapa BraLTP1 (Bra011229) [32], B. oleracea BolLTP1 (Bol018048) [60], B. napus BnaLTP1 (AY208878), and B. napus BnaLTP2 (KM062522). (B) T-DNA region of the BnaLTP1 overexpression construct containing BnaLTP1 driven by the CaMV 35S promoter. LB = Left border, RB = Right border, poly A = poly A terminator, nptII = kanamycin resistance, NOS Ter = nopaline synthase terminator, NOS Pro = nopaline synthase promoter. (C) Analysis of BraLTP1 mRNA levels in 10-week-old wild type (WT) and T0 35S::BraLTP1 transgenic plants by qRTPCR. Transgenic plants include BraLTP1-1, -6, -7, -19, -20, -21, -22, -28, -29, -32, -34 and -39. Standard errors were derived from three repeated experiment for the expression levels of each plants.

Mentions: The coding sequence of BraLTP1 was amplified from B. rapa accession Chiifu genomic DNA using primers designed to the published B. rapa sequence Bra011229 (http://brassicadb.org/brad/index.php) [32]. Primers were as follows: BraLTP1-F: 5′- GAGCTCACAACTTCCTTCAAAGCCACA-3′ and BraLTP1-R: 5′-GGATCCCAAACCTCATGGCACAATGTA-3′, containing 5′ restriction enzyme sites for SacI and BamHI respectively. PCR was carried out in 50 µL, with 50 ng DNA, 0.4 mM dNTPs, 0.2 µM each primer, 0.5 U LA Taq (TaKaRa, Japan) and 1×LA Taq buffer II (TaKaRa, Japan). Conditions were: 94°C for 3 min, 30 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. PCR product was checked by gel electrophoresis and target fragment was recovery and purified. The purified PCR product was cleaved using SacI and BamHI, and ligated between the CaMV 35S promoter and a terminal poly A sequence in the vector PBI121s (Fig. 1B), derived by modifying the multiple cloning site and deleting the GUS gene of PBI121 [33]. Positive clones by PCR using the above gene specific primers were chosen and sequenced (sangon company of Shanghai, China) to make sure they were correct. Standard molecular techniques [34], [35] were used for DNA manipulation.


BraLTP1, a lipid transfer protein gene involved in epicuticular wax deposition, cell proliferation and flower development in Brassica napus.

Liu F, Xiong X, Wu L, Fu D, Hayward A, Zeng X, Cao Y, Wu Y, Li Y, Wu G - PLoS ONE (2014)

Basic protein characteristics of BraLTP1 and vector construction.(A) Analysis of the deduced amino acid sequences of BraLTP1 with its homologous sequences in other cruciferae; variable sites (dark grey)the nsLTP-like conserved 8 CM domain (light gray) with conserved cysteine residues (asterisks) and putative extracellular secretory signals (underlined). Sequences are from Arabidopsis thaliana AtLTP1 (AT4g30880), B. rapa BraLTP1 (Bra011229) [32], B. oleracea BolLTP1 (Bol018048) [60], B. napus BnaLTP1 (AY208878), and B. napus BnaLTP2 (KM062522). (B) T-DNA region of the BnaLTP1 overexpression construct containing BnaLTP1 driven by the CaMV 35S promoter. LB = Left border, RB = Right border, poly A = poly A terminator, nptII = kanamycin resistance, NOS Ter = nopaline synthase terminator, NOS Pro = nopaline synthase promoter. (C) Analysis of BraLTP1 mRNA levels in 10-week-old wild type (WT) and T0 35S::BraLTP1 transgenic plants by qRTPCR. Transgenic plants include BraLTP1-1, -6, -7, -19, -20, -21, -22, -28, -29, -32, -34 and -39. Standard errors were derived from three repeated experiment for the expression levels of each plants.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196963&req=5

pone-0110272-g001: Basic protein characteristics of BraLTP1 and vector construction.(A) Analysis of the deduced amino acid sequences of BraLTP1 with its homologous sequences in other cruciferae; variable sites (dark grey)the nsLTP-like conserved 8 CM domain (light gray) with conserved cysteine residues (asterisks) and putative extracellular secretory signals (underlined). Sequences are from Arabidopsis thaliana AtLTP1 (AT4g30880), B. rapa BraLTP1 (Bra011229) [32], B. oleracea BolLTP1 (Bol018048) [60], B. napus BnaLTP1 (AY208878), and B. napus BnaLTP2 (KM062522). (B) T-DNA region of the BnaLTP1 overexpression construct containing BnaLTP1 driven by the CaMV 35S promoter. LB = Left border, RB = Right border, poly A = poly A terminator, nptII = kanamycin resistance, NOS Ter = nopaline synthase terminator, NOS Pro = nopaline synthase promoter. (C) Analysis of BraLTP1 mRNA levels in 10-week-old wild type (WT) and T0 35S::BraLTP1 transgenic plants by qRTPCR. Transgenic plants include BraLTP1-1, -6, -7, -19, -20, -21, -22, -28, -29, -32, -34 and -39. Standard errors were derived from three repeated experiment for the expression levels of each plants.
Mentions: The coding sequence of BraLTP1 was amplified from B. rapa accession Chiifu genomic DNA using primers designed to the published B. rapa sequence Bra011229 (http://brassicadb.org/brad/index.php) [32]. Primers were as follows: BraLTP1-F: 5′- GAGCTCACAACTTCCTTCAAAGCCACA-3′ and BraLTP1-R: 5′-GGATCCCAAACCTCATGGCACAATGTA-3′, containing 5′ restriction enzyme sites for SacI and BamHI respectively. PCR was carried out in 50 µL, with 50 ng DNA, 0.4 mM dNTPs, 0.2 µM each primer, 0.5 U LA Taq (TaKaRa, Japan) and 1×LA Taq buffer II (TaKaRa, Japan). Conditions were: 94°C for 3 min, 30 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. PCR product was checked by gel electrophoresis and target fragment was recovery and purified. The purified PCR product was cleaved using SacI and BamHI, and ligated between the CaMV 35S promoter and a terminal poly A sequence in the vector PBI121s (Fig. 1B), derived by modifying the multiple cloning site and deleting the GUS gene of PBI121 [33]. Positive clones by PCR using the above gene specific primers were chosen and sequenced (sangon company of Shanghai, China) to make sure they were correct. Standard molecular techniques [34], [35] were used for DNA manipulation.

Bottom Line: Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type.This was consistent with altered expression of a a number of ABC model genes related to flower development.Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.

ABSTRACT
Plant non-specific lipid transfer proteins (nsLTPs) constitute large multigene families that possess complex physiological functions, many of which remain unclear. This study isolated and characterized the function of a lipid transfer protein gene, BraLTP1 from Brassica rapa, in the important oilseed crops Brassica napus. BraLTP1 encodes a predicted secretory protein, in the little known VI Class of nsLTP families. Overexpression of BnaLTP1 in B. napus caused abnormal green coloration and reduced wax deposition on leaves and detailed wax analysis revealed 17-80% reduction in various major wax components, which resulted in significant water-loss relative to wild type. BnaLTP1 overexpressing leaves exhibited morphological disfiguration and abaxially curled leaf edges, and leaf cross-sections revealed cell overproliferation that was correlated to increased cytokinin levels (tZ, tZR, iP, and iPR) in leaves and high expression of the cytokinin biosynthsis gene IPT3. BnaLTP1-overexpressing plants also displayed morphological disfiguration of flowers, with early-onset and elongated carpel development and outwardly curled stamen. This was consistent with altered expression of a a number of ABC model genes related to flower development. Together, these results suggest that BraLTP1 is a new nsLTP gene involved in wax production or deposition, with additional direct or indirect effects on cell division and flower development.

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