7 research outputs found
Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display
Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display
Cotton fibers are differentiated, non-dividing cells that originate
from the epidermal layer of developing ovules. To identify genes
involved in cotton fiber development, we performed non-radioactive
differential display reverse transcriptase PCR (DDRT-PCR) on the
purified mRNA. This technique was tested on mRNA isolated from five
different developmental stages of cotton fiber including 0, 5, 10, 15
and 20 DPA (days after pollination). The mRNA purified from total RNA
was reversibly transcribed using three anchored oligo-dT primers.
Polymerase chain reaction (PCR) amplification of each cDNA preparation
was carried out in combination with seven arbitrary primers. The
amplified products were resolved on 1% agarose gel containing ethidium
bromide. DNA was extracted from seventeen differentially expressed
bands and cloned in pTZ57R/T vector. The sequencing and BLAST search
analysis indicated that 12 of the differentially expressed genes
matched the previously characterized genes, while 3 of them matched the
uncharacterized sequences of cotton fiber expressed sequence tags
(ESTs) reported previously to be associated with cotton fiber and 2 of
the clones had homology with putative proteins. The technique can be
used to efficiently identify differentially expressed genes and can be
expanded to large scale studies by increasing the number of random
decamers
Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers
The Calotropis procera seed fibers provide an excellent model system
to study the genes involved in fiber elongation, fineness and strength.
Expansins constitute one of the important gene families involved in
plant cell expansion and other cell wall modification processes. Four
homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4
were isolated from the cDNA library obtained from fast growing
Calotropis procera fibers. These homologs represented typical Expansin
A family. Each of them had two conserved domains including GH45 like
domain and the putative polysaccharide binding domain. The deduced
amino acid sequences of the homologs indicated three conserved motifs:
i) eight cysteine residues at N-terminus, ii) four tryptophan residues
at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the
center of the sequence. The presence of N-terminal signal peptide
consisting of hydrophobic amino acids and a transmembrane region in all
these expansin isoforms suggests their cotranslational insertion into
the endoplasmic reticulum and then transportation to the cell wall by
secretory pathway. The relative quantification of the four expansins in
root, stem, fiber and leave tissues indicated that the transcripts of
CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these
tissues. The lowest transcription of all the four Expansin A isoforms
was observed in elongating roots indicating that root tissue might be
having specific expansins other than those confined to air grown
organs
Identification of differentially expressed genes in developing cotton fibers (Gossypium hirsutum L) through differential display
Cotton fibers are differentiated, non-dividing cells that originate
from the epidermal layer of developing ovules. To identify genes
involved in cotton fiber development, we performed non-radioactive
differential display reverse transcriptase PCR (DDRT-PCR) on the
purified mRNA. This technique was tested on mRNA isolated from five
different developmental stages of cotton fiber including 0, 5, 10, 15
and 20 DPA (days after pollination). The mRNA purified from total RNA
was reversibly transcribed using three anchored oligo-dT primers.
Polymerase chain reaction (PCR) amplification of each cDNA preparation
was carried out in combination with seven arbitrary primers. The
amplified products were resolved on 1% agarose gel containing ethidium
bromide. DNA was extracted from seventeen differentially expressed
bands and cloned in pTZ57R/T vector. The sequencing and BLAST search
analysis indicated that 12 of the differentially expressed genes
matched the previously characterized genes, while 3 of them matched the
uncharacterized sequences of cotton fiber expressed sequence tags
(ESTs) reported previously to be associated with cotton fiber and 2 of
the clones had homology with putative proteins. The technique can be
used to efficiently identify differentially expressed genes and can be
expanded to large scale studies by increasing the number of random
decamers
Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers
The Calotropis procera seed fibers provide an excellent model system
to study the genes involved in fiber elongation, fineness and strength.
Expansins constitute one of the important gene families involved in
plant cell expansion and other cell wall modification processes. Four
homologs of Expansin A gene i.e. CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4
were isolated from the cDNA library obtained from fast growing
Calotropis procera fibers. These homologs represented typical Expansin
A family. Each of them had two conserved domains including GH45 like
domain and the putative polysaccharide binding domain. The deduced
amino acid sequences of the homologs indicated three conserved motifs:
i) eight cysteine residues at N-terminus, ii) four tryptophan residues
at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the
center of the sequence. The presence of N-terminal signal peptide
consisting of hydrophobic amino acids and a transmembrane region in all
these expansin isoforms suggests their cotranslational insertion into
the endoplasmic reticulum and then transportation to the cell wall by
secretory pathway. The relative quantification of the four expansins in
root, stem, fiber and leave tissues indicated that the transcripts of
CpEXPA1, CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these
tissues. The lowest transcription of all the four Expansin A isoforms
was observed in elongating roots indicating that root tissue might be
having specific expansins other than those confined to air grown
organs