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Research Article
Open Access
Abstract Genome wide analysis of heat shock factor (Hsf) genes was carried out in pigeonpea (Cajanus cajan) in order to
understand their structure and function. A total of 23 Hsfs were predicted using FGENESH and labeled as CcHsf. Out of the 23 genes,
14 unique sequences were selected and characterized for their presumed structures such as protein domain and motif organization.
The phylogenetic relationships and expression profiling of CcHsf genes under heat-stress was studied. Phylogenetic analysis
showed that CcHsf genes were distributed into e i g h t groups. In this study, classes A, B and C were further subdivided into
subclasses such as A1, A2, A3, A4, A5, A6, A8, A9, B 1 , B2, B3, B4 and C1. Expression profiling of all 14 genes was carried out by
semi-quantitative PCR, among which CcHsfA-1d and CcHsfA-2 were observed to be highly upregulated during h ea t -st re ss .
Relative quantification with qRT-PCR s h o w e d that CcHsfA-1d is upregulated 2-6 hrs after heat-stress indicating its significant role
as an early response factor. Our study provides a glimpse of the Hsf gene family in pigeonpea and this information can be utilized to
gain more insight into the heat-response mechanism in pigeonpea.
Keywords Domain; Glycine max; Heat shock factor; Motif; Pigeonpea; Phylogenetics
Abbreviations: AHA - Aromatic/ Hydrophobic /Acidic; Hsf - Heat Shock Factors; IRRK- Isoleucine, Arginine, Arginine,
Lysine; NLS - Nuclear Localization Signal; NES - Nuclear Export Signal
Introduction
2 Results
Sequence
Tm
R AACCCCACTATGCCCTTGC
53
F GCAGGTGAAAGAAGAGGTGGA 54
R TCAGGAACGGTGGTGGAC
53
F TTCAAACCTCTCTCCGCAAC
52
R GTCGGTGGATGGGTCTTCA
53
F ACAGCCTATGGAAGGGTTGC
54
R CAGCAGAAAACTTGTGAGAATCC53
F TGGAGCCAAACTTGCGATAG
52
R GCGAACGAAGCTTGAGAAATTG 53
Gene
CcHsfA-1d F TCATTGGGGGTTATTGGATCA 62
Sequence
Tm
Tubulin-A F TGCCACCATCAAGACTAAGAGG 60
R GCTGGACCTCATTCCCTTTG 62
R ACCACCAGGAACAACAGAAGG60
Table 3 Predicted gene analysis
SL.No
Gene
Molecular weight(Da)
pI
CcHsf B-3
231
26740.1
9.08
CcHsf A-1b
436
48602.2
5.03
CcHsf A-4a
402
46005.0
5.05
CcHsf A-9
426
48470.6
5.87
CcHsf A-6b
276
32123.3
5.64
CcHsf B-2a
381
41740.2
5.27
CcHsfA-8-like
175
19277.7
5.31
CcHsf A-1d
561
62434.3
5.17
CcHsf A-2
366
41361.0
4.91
10
CcHsf A-6b-like
353
41087.1
5.25
11
CcHsf A-8
438
50363.0
5.17
12
CcHsf A-1d-like
416
45821.1
4.81
13
CcHsf A-4a-like
388
44374.5
5.12
14
CcHsf B-2a-like
257
28819.5
9.24
Gene
CcHsfB-3
CcHsfA-1b
CcHsfA-4a
CcHsfA-6b
CcHsfA-6b-like
CcHsfA-2
CcHsfA-4a
Figure 2a Phylogenetic relationships among pigeonpea, soybean, medicago and Arabidopsis using MEGA 5., iTOL (Interactive
Tree Of Life)
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3 Discussion
In this study, 14 CcHsf genes were identified and
characterized from the draft pigeonpea genome. All
predicted CcHsf genes displayed conserved Hsf-type
DNA binding domain (DBD) indicating that it is the
most conserved among the different domains in Hsf.
This is in agreement with Arabidopsis Hsfs where the
DNA Binding Domain is highly conserved (IRRK--),
(Nover et al., 2001; Scharf et al., 1993, 2012). Both
NLS and NES, which are important for maintaining
the intracellular distribution of the Hsfs between the
nucleus and cytoplasm (Lyck et al., 1997, Baniwal et
al., 2004) were also found in pigeonpea proteins. The
multiple alignment and distribution of signature
domains indicated the presence of atleast 14 genes in
pigeonpea. Peculiar alignment was seen for A-d and
A-1d like indicating low level of homology among
other genes. This is strange since the prediction was
based on legume database hsf genes. The A-1d like
Figure 4 QRT-PCR of CcHsf A-1d gene expression (Bud from Asha cv.) during heat stress under 2-24 hr interval
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