Lipid transfer proteins in coffee: isolation of Coffea orthologs, Coffea arabica homeologs, expression during coffee fruit development and promoter analysis in transgenic tobacco plants.
The aim of the present study was to perform a genomic analysis of non-specific lipid-transfer proteins (nsLTPs) in coffee. Several nsLTPs-encoding cDNA and gene sequences were cloned from Coffea arabica and Coffea canephora species. In this work, their analyses revealed that coffee nsLTPs belong to Type II LTP characterized under their mature forms by a molecular weight of around 7.3 kDa, a basic isoelectric points of 8.5 and the presence of typical CXC pattern, with X being an hydrophobic residue facing towards the hydrophobic cavity. Even if several single nucleotide polymorphisms were identified in these nsLTP-coding sequences, 3D predictions showed that they do not have a significant impact on protein functions. Northern blot and RT-qPCR experiments revealed specific expression of Type II nsLTPs-encoding genes in coffee fruits, mainly during the early development of endosperm of both C. arabica and C. canephora. As part of our search for tissue-specific promoters in coffee, an nsLTP promoter region of around 1.2 kb was isolated. It contained several DNA repeats including boxes identified as essential for grain specific expression in other plants. The whole fragment, and a series of 5' deletions, were fused to the reporter gene β-glucuronidase (uidA) and analyzed in transgenic Nicotiana tabacum plants. Histochemical and fluorimetric GUS assays showed that the shorter (345 bp) and medium (827 bp) fragments of nsLTP promoter function as grain-specific promoters in transgenic tobacco plants.
Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, CP 02372, Brasília, DF, 70770-917, Brazil.
Michelle G
MG
Cotta
Leila M G
LM
Barros
Juliana D
JD
de Almeida
Fréderic
F
de Lamotte
Eder A
EA
Barbosa
Natalia G
NG
Vieira
Gabriel S C
GS
Alves
Felipe
F
Vinecky
Alan C
AC
Andrade
Pierre
P
Marraccini
D000595:Amino Acid Sequence; D052179:Antigens, Plant / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D003069:Coffee / Q000235:genetics*; D017931:DNA Primers; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D052179:Antigens, Plant; D002352:Carrier Proteins; D003069:Coffee; D017931:DNA Primers; D010940:Plant Proteins; C511920:lipid transfer proteins, plant