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International Journal of Agriculture and Crop Sciences.
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IJACS/2015/8-5/706-712
ISSN 2227-670X ©2015 IJACS Journal
Isolation of a suitable endogenous “killer” gene
callase or β-1, 3-glucanase for inducing male sterility
in jute (Corchorus olitorius)
Monami Chakraborty, Soumitra K. Sen*
Adv. Lab. for Plant Genetic Engineering, Advanced Technology Development Center, Indian Institute of
Technology Kharagpur, India
*
Corresponding author email: [email protected]
ABSTRACT: Jute (Corchorus olitorius) is an important natural fiber producing plant of commercial
importance. Hybrid breeding is an important strategy to improve crop production. Among several
methods. Male sterility is basically two types-cytoplasmic and nuclear. In nuclear male sterility an anther
specific promoter can be used to drive a male sterility inducing “killer” gene in spatio-temporal manner.
Callase is an important male sterility related gene that lyase carbohydrate. The product of the callase
gene, β-1, 3-glucanase enzyme break down the callose (glucan) layer that surrounds the immature pollen
in tetrad and release pollens which play important role in fertilization in the plant. This strategy of male
sterility has been proved to be an effective one to generate hybrid plant. In this study a suitable
endogenous “Killer” gene (β-1, 3-glucanase) for inducing male sterility in jute was isolated (NCBI
accession no AGW45300.1) by degenerate PCR, 5'-3 ' RACE and genome walking experiments. In silico
analysis showed that the predicted CDS of β-1, 3-glucanase gene is 1020 bp (2 exons of 93 bp and 927
bp and one intron of 857 bp) with amino acid residues of 340 and approximate molecular wt. of ~37.4
kDa. The full length predicted protein sequence showed more than 77% sequence similarity with other
reported β-1, 3-glucanase gene of plant and posses conserved domain Glycosyl hydrolases (like other
reported beta-1, 3-glucanase ) and other functional motifs required for its function.
Key words: Jute (Corchorus olitorius), Male sterility, β-1, 3-glucanase, RACE, Genome walking
INTRODUCTION
Jute has been attracted attention from long time for the production of natural fiber of commercial
importance. Fiber of jute is mostly used for spinning of rough quality fabrics such as heavy duty clothing, home
furnishings and carpet backing. In India “Tossa” jute (Corchorus olitorius) is a variety thought to be native, and is
also the world's top producer. Research focus on improved productivity of jute is an important proposition. Hybrid
breeding is one of the best methods to increase productivity in crop species. Hybrid plants have the advantages of
higher yield and better disease resistance than their parents. The male sterility system becomes an effective tool
for hybrid seed production. Male sterile plants are useful for the production of hybrid plants by sexual hybridization.
Producing a hybrid plant entails ensuring that the female parent does not self fertilize. Several techniques, viz.,
mechanical, chemical, and genetic, have been proposed for preventing self pollination. Among these methods
mechanical and chemical methods have some disadvantages. So the genetic methods are mostly used for
generation of male sterile plant. Cytoplasmic male sterility and nuclear male sterility are two common method
utilized for the purpose of male sterility. In nuclear male sterility the use of anther-specific genes or their promoters
to disrupt the normal production of pollen grains is a proven approach. An anther specific promoter can be used to
drive a male sterility inducing “killer” gene in a spatio-temporal manner. Male sterility gene functions include those
that code for lytic enzymes, including those that lyase proteins, nucleic acids and carbohydrates.
Glucanases are the enzymes which break down carbohydrates. Callose is a polymer of β-1, 3-glucanases
with some β-1, 6-branches, which plays an important role in plant pollen development. In male gametogenesis, the
processes of callose synthesis and degradation are highly regulated. At the onset of meiosis, a secondary callose
wall is deposited between the primary wall and plasma membrane of the pollen mother cells (PMCs). This
temporary callose wall is believed to prevent the cohesion and fusion of the PMCs and it may function as a
Intl J Agri Crop Sci. Vol., 8 (5), 706-712, 2015
molecular filter that protects the developing pollen cells from the surrounding diploid tissues (Heslop-Harrison and
Mackenzie 1967). It can also provide a flexible wall that may help prevent premature swelling and bursting of the
microspores. In addition, the callose wall can provide a template or mold for formation of the exine wall of pollen
grains (Worrall et al. 1992; Dong et al. 2005). The basis of the use of glucanase as the sterility causing agent lies in
the fact that mis-timing of the appearance of callase activity can be associated with certain types of male sterility
(Warmke and Overman 1972). The product of the callase gene, β-1, 3-glucanase enzyme break down the callose
(glucan) layer, surroundings the immatured pollen in tetrad forms and release matured pollens which play important
role in fertilization in the plant. Expressing the target “killer” gene callase under the anther specific gene promoter in
early meiotic stage of the microsporogenesis, would result in the release of immature pollens that could lead to
male sterility. Therefore to generate male sterility in jute following this approach we try to isolate callase or β-1, 3glucanase gene from jute. Previously no β-1, 3-glucanase gene was reported from C. olitorius. In this study we
have isolated full length β-1, 3-glucanase gene for inducing male sterility in jute.
MATERIALS AND METHODS
Isolation of genomic DNA from anther of Jute plant
The genomic DNA isolation was carried out following cetyltriethyl ammonium bromide (CTAB) extraction
method described by Doyle and Doyle, 1990. The quantity of the DNA was measured at 260 nm wavelength in
spectrophotometer (Hitachi, U 2001 spectrophotometer) and the quality of the isolated genomic DNA was verified
through restriction digestion followed by agarose gel (0.8%) electrophoresis.
PCR and cloning of partial CDS of β -1, 3- glucanase gene of Jute
For amplification of the β-1, 3-glucanase gene and A9 gene from Jute, the β-1, 3-glucanase protein
sequences were aligned (using software clustalW) from the following plants: Arabidopsis, Brassica, Nicotiana,
Gossypium, Glycine, Hevea and Sesbania. The Degenerate primers were designed from the conserved regions of
the aligned proteins. PCR amplification was carried out by Taq DNA polymerase using genomic DNA as template.
The amplified PCR products were cloned into the TA cloning vector (Invitrogen) and sequenced. The sequence
analysis was done by NCBI Blast program and Jellyfish software.
Total RNA isolation from anther
Isolation of total RNA from anther of Jute plant at particular developmental stage was carried out by the
RNesay Plant Mini Kit (Qiagen), and the isolated RNA was treated with RNase-Free DNase (Qiagen) to eliminate
the contaminating genomic DNA. Isolated total RNA was analysed through agarose gel electrophoresis and
spectrophotometric absorbance at 260/280 nm wavelengths for quality and quantity assessment of the RNA,
respectively.
RACE (rapid amplification of cDNA ends) and PCR amplification
For the 3' RACE experiment, a poly (A) tail was synthesized using poly (A) polymerase at the 3' ends
(Invitrogen). The polymerization reaction was carried out with 5 µg of RNA in a final volume of 50 mL containing 50
mM Tris–HCl (pH 7.9), 10 mM MgCl2, 2.5 mM MnCl2, 50 mM NaCl, 250 mM ATP, 500 µg mL-1 BSA and 5 U poly
(A) polymerase for 2 h at 37°C. Polyadenylated RNA was purified by phenole–CHCl3 extraction and ethanol
precipitation overnight at -70°C. Subsequently, 1st strand cDNA was synthesized using AOT (oligo dT adapter)
primer at 48°C for 1 h. PCR was carried out with polyadenylated cDNA as template using the 50 gene-specific
primer designed on the basis of N-terminal sequence of the peptide and 3' RACE-AMP primer (provided in kit) that
annealed to the poly (A) tail. PCR was done under the following thermal profile: initial denaturation at 94°C for 4
min, followed by 30 cycles of 94°C/30s, 55°C/30s, 72°C/30s and a final extension at 72°C for 5 min with Taq DNA
polymerase (Roche). The 3'RACE PCR product was resolved in 1.0% agarose gel, eluted using quick gel
extraction kit (QIAGEN) and cloned into pCR2.1-TOPO vector. Recombinant plasmid pCR2.1-TOPO10/RACE PCR
product was sequenced using M13 forward and reverse primer with Bigdye terminator kit (ABI). Similarly 5' RACE
experiment was carried out according to manufcturers protocol.
Genome walking
Genome walking was carried out by using the Clontech Genome walking kit. Digestion of genomic DNA
and adaptor ligation was carried out according to manufacturer’s instructions. For PCR reactions, two gene specific
3' primers were designed from both CDS of the callase gene and A9 gene. Primary and secondary PCR reactions
were carried out according to vendor’s protocols using the adaptor primers (kit provided) and respective gene
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specific primers. The amplified strong PCR product in secondary PCR was cloned into TA cloning vector
(Invitrogen) and sequenced. The bioinformatics analysis of the sequences was carried out using NCBI Blast and
Jellyfish software.
RESULTS
A PCR experiment was carried out using degenerate primers to isolate the callase (β-1, 3- glucanase)
gene fragment using genomic DNA of jute as template. About 489 bp amplified DNA fragment was cloned in TA
vector. After sequencing, possible ORFs (having amino acid length >100) were identified by Jellyfish 3.3.1
software. An ORF containing 163 amino acids was identified (Figure 1) and a protein BLAST was carried out. The
protein BLAST results revealed up to 80% sequence homology with the other known β-1, 3- glucanase from related
crop species. Thus it was confirmed that the amplified sequence was similar with β -1, 3- glucanase gene fragment.
To isolate full length β -1, 3-glucanase gene two strategies were undertaken: (a) 5' and 3' RACE experiment from
RNA to isolate coding DNA sequence consisting of exons obtained after splicing, (b) from genomic DNA using
chromosome walking strategy to unravel the complete genomic level organization of the gene including introns, if
any.
For isolation of the 3' part of the β-1, 3-glucanase gene, the 3' RACE experiment was carried out. The total
RNA was isolated from the anther of jute. 1st strand cDNA was synthesized using oligodT primer. The RT-PCR was
carried out using gene specific primer [SP5(1)] and oligodT primer. The 170 bp PCR amplified fragment was cloned
and sequenced. The bioinformatic analysis of the nucleotide sequence, as described above, confirmed the isolation
of 3' part of the β-1, 3-glucanase gene. 5' RACE was also carried out to isolate the 5' portion of the β-1, 3glucanase gene fragment. For this, vendor’s protocol was followed and after polyadenylation, RT-PCR was carried
out using 3' gene specific primers (SP1) and oligodT primer. The amplified DNA fragment of around 1.0 kb was
cloned and sequenced. The sequence analysis confirmed that the amplified sequence was the 5' portion of the β-1,
3-glucanase gene fragment.
A second round of genome walking was carried out further to isolate the remaining 5' portion of the so far
isolated callase gene fragment. A strong amplification band of ~1 kb size obtained after secondary PCR. The
amplified fragment was cloned and sequenced. The sequence analysis confirmed the isolation of upstream portion
of the same β-1, 3-glucanase gene fragment. Interestingly, bioinformatics analysis also revealed the presence of
an intron of 851 bp just after the 1st exon. The 1st exon was also found to code for a transmembrane domain as
revealed by SOSUI and TMHMM software analysis (data not shown). Thus the complete coding sequence of the
beta-1, 3-glucanase could be deciphered along with an intervening intron.
Previously done sequence analysis of 5' and 3' RACE fragments by BLAST analysis confirmed the identity
of β-1, 3-glucanase gene sequence. About 1kb fragment obtained by genome walking and 5' RACE showed
sequence homology. However, additional DNA sequence was obtained for genome walking. Sequence alignment
of all the fragments finally derived converged full length genomic DNA and cDNA of β-1, 3-glucanase gene of jute.
Alignment revealed the presence of one intron in the genomic organization of the β-1, 3-glucanase gene. Region
108 to 200 showed 1st exon of 93 bp, region 201 to 1051 showed intron of 851 bp and region 1052 to 1978 showed
2nd exon of 927 bp (Figure 2). Sequence was submitted to GenBank with NCBI accession no AGW45300.1. The
predicted CDS of β-1, 3-glucanase gene is 1020 bp with amino acid residues of 340 and approximate molecular wt.
of ~37.4 kDa. The full length predicted protein sequence showed more than 77% sequence similarity with other
reported β-1, 3- glucanase gene of plant.
To verify in silico the β-1, 3-glucanase specific conserved motifs in the newly cloned isoforms, the amino
acid sequences of β-1, 3-glucanase of jute was aligned with homologous sequences of β-1, 3-glucanase obtained
from other plant using CLUSTALW (Figure 3). The region with remarkable sequence identity was observed among
all β-1, 3-glucanase was the glycosylhydrolase domain with signature motif G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}.
To verify the presence of characteristic membrane bound domain amino acid sequence was analyzed by KyteDoolittle hydropathy plots (Figure 4). It was revealed from the hydrophobicity plot that amino acid sequence 13 to
33 posses membrane integration domain i.e. in the 1st exon position like other protein. Phylogenetic analysis
showed the β-1, 3-glucanase gene of jute is closely similar to other reported β-1, 3-glucanse gene from related
plants.
DISCUSSION
The pollen development involves a number of events, commencing at the end of meiosis with the formation
of tetrads of haploid microspores (Scott et al., 2004). Individual microspores in each tetrad are surrounded by thick
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callose composed of a β-1, 3-glucan, between the primary cell wall and the plasma membrane (Eschrich and
Currier 1964). At the end of meiosis, tetrads of haploid microspores are freed into the locule (Steiglitz and Stern
1973). The anther-specific expression of β-1, 3-glucanase mRNA and protein is detectable from meiosis to the free
microspore stages in many plant species, and these enzymes are thought to participate in tetrad dissolution during
pollen development (Hird et al., 1993; Bucciaglia and Smith 1994). The expression of Brassica napus and
Arabidopsis thaliana A6 gene is tapetum specific and temporally correlated with the expression of callase activity
(Hird et al. 1993). A tobacco anther β-1, 3-glucanase Tag1 is also expressed exclusively in the tapetum and has a
callase-like expression pattern (Bucciaglia and Smith 1994). Several petunia mutants with perturbed callose wall
formation and degradation have been characterized, and it has been suggested that the timing of callose wall
formation and degradation is important for proper microsporegenesis and pollen development (Warmke and
Overman 1972). In the Arabidopsis callose synthase mutant cals5 normal callose deposition does not occur in
meiocytes, tetrads, microspores, and mature pollen. Consequently, the pollen exine wall is not formed properly,
and the mutant exhibits male sterility (Dong et al. 2005). However, little information is currently available on the
function of β-1, 3- glucanases that are expressed in the anthers of jute. In future expression of β-1, 3- glucanases
of jute under the regulation of jute anther specific promoter in tapetum (homologue to A9 gene promoter of A.
thaliana) may results dissolution of the callose wall prematurely in early stage of meiosis, causing male sterility.
In conclusion, a suitable endogenous “Killer” gene (β-1, 3-glucanase) for inducing male sterility in jute was
isolated by degenerate PCR, 5'-3 ' RACE and genome walking experiments. In silico analysis showed that the
predicted CDS of β-1, 3-glucanase gene is 1020 bp (2 exons of 93 bp and 927 bp and one intron of 857 bp) with
amino acid residues of 340 and approximate molecular wt. of ~37.4 kDa. The full length predicted protein
sequence showed more than 77% sequence similarity with other reported β-1, 3-glucanase gene of plant and
posses conserved domain Glycosyl hydrolases (like other reported beta-1,3-glucanase ) and other functional motifs
required for its function.
Figure 1. (a) 1% agarose gel showing the PCR amplified DNA fragment of β-1, 3-glucanase gene, (b) Cloning of PCR product in
pTZ57R/T (TA) cloning vector for sequencing of the amplicon, (c) Partial CDS sequence obtained after processing the raw
sequence with corresponding amino acid sequence. Red underline region heighted the region from where degenerate primers
were designed.
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Figure 2. Full length β-1, 3- glucanase gene and the exon regions of same gene was alligned. Yellow highlighted region display
exon regions, whereas white region display only intron part.
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Figure 3. CLUSTALW alignment of β-1, 3-glucanase of C. olitorious with reported β-1, 3-glucanase of other plants. Black box
highlighted region indicating the presence of characteristic Glycosyl hydrolases motif required for its “callase” activity
Figure 4. Kyte-Dollitle plot of transmembrane domain assay
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ACKNOWLEDGEMENTS
Authors thank Dr. Anirban Chakraborty for his technical help and Dr. A. Basu for his cooperation. Grant
support to Advanced Laboratory for Plant Genetic Engineering from Indian Council of Agricultural Research is
thankfully acknowledged.
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