6 research outputs found
Physiological responses induced by phospholipase C isoform 5 upon heat stress in Arabidopsis thaliana
Plant’s perception of heat stress involves several pathways and signaling molecules, such as phosphoinositide, which is derived from structural membrane lipids phosphatidylinositol. Phospholipase C (PLC) is a well-known signaling enzyme containing many isoforms in different organisms. In the present study, Phospholipase C Isoform 5 (PLC5) was investigated for its role in thermotolerance in Arabidopsis thaliana. Two over-expressing lines and one knock-down mutant of PLC5 were first treated at a moderate temperature (37 °C) and left for recovery. Then again exposed to a high temperature (45 °C) to check the seedling viability and chlorophyll contents. Root behavior and changes in 32Pi labeled phospholipids were investigated after their exposure to high temperatures. Over-expression of PLC5 (PLC5 OE) exhibited quick and better phenotypic recovery with bigger and greener leaves followed by chlorophyll contents as compared to wild-type (Col-0) and PLC5 knock-down mutant in which seedling recovery was compromised. PLC5 knock-down mutant illustrated well-developed root architecture under controlled conditions but stunted secondary roots under heat stress as compared to over-expressing PLC5 lines. Around 2.3-fold increase in phosphatidylinositol 4,5-bisphosphate level was observed in PLC5 OE lines upon heat stress compared to wild-type and PLC5 knock-down mutant lines. A significant increase in phosphatidylglycerol was also observed in PLC5 OE lines as compared to Col-0 and PLC5 knock-down mutant lines. The results of the present study demonstrated that PLC5 over-expression contributes to heat stress tolerance while maintaining its photosynthetic activity and is also observed to be associated with primary and secondary root growth in Arabidopsis thaliana
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
Cloning and analysis of NBS-LRR super family of resistance (R) genes in wheat (Triticum aestivum L.)
Abstract: Resistance (R) genes containing nucleotide binding site (NBS) and leucine rich repeats (LRR) are the most prevalent types of resistance (R) genes in plants. The objective of this study was to isolate, identify and analyze resistance genes from disease (rust) and resistant wheat lines by PCR based strategy. Fifteen degenerate primers were designed from the conserved kinase-la and hydrophobic domains of known NBS-LRR type R-genes and from EST data bases. Four advanced resistant lines and one susceptible wheat line was selected from the trap nursery. Out of hundred primer combinations only seventy five primer combinations showed amplification. Twenty two primer combination showed differential banding pattern which were not present in highly susceptible Morocco, were cloned in TA based cloning vector and got them sequenced. Sizes of sequenced nucleotides were between 500bp to 1500bp. The cloned fragments showed their DNA sequence similarity to known resistance (R) genes of NBS-LRR family. These results indicate that identified genes are the valuable source to use as disease resistance genes or to screen wheat resistant germplasm against different types of rusts
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