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Differential Response of Cysteine-deficient Lentil (
Lens culinaris
Medik.) Mutants Impaired in Foliar
O
-acetylserine(thiol)-lyase
Expression
38
3.6 Relative gene expression analysis through
quantitative RT-PCR
Gene expression levels of SAT and OAS-TL of
control, two mutant lines, and segregating progeny
plants were analyzed
by quantitative reverse
transcription polymerase chain
reaction (qRT-PCR)
technique. Total RNA was isolated using the RNA
isolation kit (Chromous Biotech, Bangalore, India)
and treated with DNaseI (Chromous Biotech,
Bangalore, India) at 37°C for 30 min. The quality of total
RNA samples was determined spectrophotometrically
(Systonic, Kolkata, India) from
A
260/280 ratio and by
1% agarose gel electrophoresis. First strand cDNA
was synthesized from DNA-free intact RNA with
oligo-dT primers and with MmuLV reverse
transcriptase enzyme kit (Chromous Biotech, Bangalore,
India) following manufacturer’s instructions.
Quantitative RT-PCR of first stand cDNA was run on
ABI Step-One (Applied Biosystems, Foster City, CA)
Real Time PCR machine. Amplification was done in a
total reaction volume of 50 µl, containing template
(first strand cDNA) 2.0 µl, forward primer 2.0 µl (100
ng), reverse primer 2.0 µl (100 ng), 2X PCR SYBR
green ready mixture (Fast Q-PCR Master Mix,
Chromous Biotech, India), 25.0 µl, and DEPC water
19.0 µl. Primers for selected genes (Table 2) were
constructed by
Primer Express
TM
V. 3.0
software
(
Applied Biosystems
, Foster City, CA, USA) with
the
search of available sequence databases (http://www.
ncbi.nlm.nih.gov/Report=GenBank) and reports on
lentil (Talukdar and Talukdar, 2014), common beans
(Talukdar and Talukdar, 2013b) and
Arabidopsis
thaliana
(Han and Kim, 2006). The
qRT-PCR cycling
stages consisted of initial denaturation at
94°C (3 min),
followed by 35 cycles of 94°C (5 s), 62°C (10s),72°C
(10 s) and a final extension stage at 72°C (2 min). A
melting curve analysis was performed after every PCR
reaction to confirm the accuracy of each amplified
product. Samples for qRT- PCR were run in four
biological replicates with each biological replicate
contained the average of three technical replicates.
DEPC water for the replacement of template was used
as negative control. RT-PCR reaction mixtures were
loaded onto 2% agarose gels in TAE buffer. A 100 bp
DNA ladder was run on every gel. The mRNA levels
were normalized against a
Lens culinaris
EF1- α
as
the housekeeping gene and the relative (to control)
expression of target genes was calculated as 2
−ΔΔCt
(Livak and Schmittgen, 2001).
Table 2 Oligonucleotide primers used in qRT
-
PCR analysis of the expression of selected target genes in lentil genotypes
, a
F
-
forward,
R
-
reverse
Target genes
Primers (5ʹ → 3ʹ)
a
Amplicon (bp)
LcOAS
-
TL 1
F
-
CTCACAAGATTCAAGGGATAGGA
-
R
-
GTCATGGCTTCCGCTTCTTTC
-
409
LcOAS
-
TL 2
F
-
GGATCCGCAGTGTCTGTACCAACGAAA
-
R
-
GACGTCTCACAATTCTGGCTTCAT
-
399
LcSAT1;1
F–AGCCATACTTCCTTTATCTCTGAGTG
-
R
-
AACAGTATATGTACTCTCAGCAGTAAC
-
318
LcSAT1;2
F
-
GGACCATACTTCCTTTATCTCTGAGTG
-
R
-
CTACTAGCAATAATCAACCTTTTCATC
-
289
EF1
-
α
(House keeping) F
-
TGTCGACTCTGGGAAGTCAA
-
R
-
CTCTTTCCCTTTCAGCCTTG
-
198
Authors' contributions
Sole author DT designed both the field and lab experiments,
measured biochemical and molecular parameters, conducted
statistical analysis, read and approved the final manuscript.
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