TY - JOUR
T1 - Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala
T2 - A pulp yielding tree species
AU - Srivastava, Sameer
AU - Gupta, Ranadheer K.
AU - Arha, Manish
AU - Vishwakarma, Rishi K.
AU - Rawal, Shuban K.
AU - Kavi Kishor, P. B.
AU - Khan, Bashir M.
N1 - Funding Information:
This work was supported by grants from Council of Scientific and Industrial Research -NMITLI, India. We wish to thank Director, NCL and Head, Dept. of Genetics, OU, Hyderabad, India. SS thank CSIR-UGC for his fellowship grant. RKG and RKV thank CSIR-NMITLI for their fellowship grant. MA thank CSIR for their fellowship grants. We wish to thank Mr. V. Yadav (NCCS, Pune, India) for his sincere suggestions in protein purification. We also wish to thank Ms. T. Kamat for proofreading of the manuscript.
Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2011/2
Y1 - 2011/2
N2 - Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.
AB - Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.
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U2 - 10.1016/j.plaphy.2010.11.001
DO - 10.1016/j.plaphy.2010.11.001
M3 - Article
C2 - 21123078
AN - SCOPUS:78751704154
SN - 0981-9428
VL - 49
SP - 138
EP - 145
JO - Plant Physiology and Biochemistry
JF - Plant Physiology and Biochemistry
IS - 2
ER -