Gershom Ann Titus
Characterization of eIF4E gene in banana (Musa spp.) and analysis of its expression in relation to banana bract mosaic virus infection - Vellanikkara Department of Plant Biotechnology, College of Agriculture 2022 - 53p.
MSc
Banana and plantain (Musa spp.) are one of the most important food crops especially
in the tropics. India is the leading producer of banana in the world with a share of around 25
per cent in total output. Despite its importance, banana cultivation in India is impeded by
disease incidence and pest attack resulting in serious loss to farmers. Banana bract mosaic
disease caused by banana bract mosaic virus (family Potyviridae) is a major virus disease
affecting banana. Plant viruses need host factors for maintaining their life cycle. Eukaryotic
translation initiation factors, particularly the eukaryotic translation initiation factor 4E
(eIF4E) and its isoform are host factors essential for infection by plant viruses in the genus
Potyvirus. In many crop species, natural variation in eIF4E protein confers resistance to
potyviruses while in others, which lack natural eIF4E alleles conferring resistance, gene
editing can be applied to transfer genetic resistance. Therefore, gene editing approaches for
mutation of eIF4E gene have potential in conferring potyvirus resistance to plants. Sequence
characterization of eIF4E gene in Musa spp. is necessary for future studies on gene editing
for resistance breeding. Moreover, the expression studies of eIF4E gene in relation to
BBrMV infection will provide an insight into its probable role in disease development. In this
background, the present study entitled “Characterization of eIF4E gene in banana (Musa
spp.) and analysis of its expression in relation to banana bract mosaic virus infection” was
undertaken during the period 2019 to 2022 at the Department of Plant Biotechnology,
College of Agriculture, Vellanikkara, Thrissur, with an objective to characterize the eIF4E
gene in banana cultivar Nendran and to analyse its expression in relation to BBrMV
infection.
Initially, leaf samples were collected from Nendran banana plants from field at
Banana Research Station, Kannara, Kerala. Purification of total RNA was attempted using
TRI reagent (Sigma), Purelink Plant RNA reagent (Invitrogen) and RNeasy plant mini kit
(Qiagen). Out of the three methods, good quality RNA was obtained using RNeasy plant mini
kit (Qiagen). The total RNA was converted to cDNA using Oligo(dT)18 primer and reverse
transcriptase enzyme. This was followed by PCR amplification of the eIF4E gene using two
pairs of primers targeting overlapping regions of the gene. The PCR amplicons were
sequenced and the overlapping sequences were aligned to obtain the final contig of 853 bp
using Cap3 software. The BLASTN analysis revealed that the sequence showed 99.06 %
identity with 99 % query coverage with predicted sequence of Musa acuminata subsp.
malaccensis eukaryotic translation initiation factor 4E-1 (LOC103981303) (Accession no.
XM_009397982.2). The BLASTX search against NCBI protein database revealed that the
sequence showed 99.58 % identity with predicted protein sequence of eIF4E-1 in Musa
acuminata subsp. malaccensis (Accession no. XP_009396257.1). Five exons were identified
in the coding sequence. The amino acid sequence of the eIF4E gene was deduced from the
nucleotide sequence using ExPASy translate tool. Eight open reading frames were identified
using ORF finder. The multiple sequence alignment and phylogenetic analysis showed that
the Nendran eIF4E gene was more related to sequence of eIF4E gene of Musa acuminata
subsp. malaccensis.
Quantitative real-time PCR analysis was performed to study the differential
expression of eIF4E gene in banana cultivar Nendran under healthy and diseased conditions.
Five banana plants showing typical symptoms of BBrMV and a symptomless banana plant
were initially identified from the field and later confirmed for the presence and absence of
virus respectively based on diagnostic RT-PCR with BBrMV coat protein gene specific
primers. For gene expression study, two samples each were collected from the identified
diseased and healthy plants. One pair of primer each for banana eIF4E gene (test gene) and
actin gene (endogenous control) was designed using Primer3 software for the qRT-PCR
study. The real time PCR reaction based on SYBR Green chemistry was carried out for
quantification of expression of eIF4E gene in diseased and healthy Nendran banana. The
relative expression levels of the gene was normalised with the expression of endogenous
control actin gene following 2-∆∆Ct method. The diseased banana plants showed 1.48 to 16.92
fold increase in the expression of eIF4E gene when compared to the healthy plants.
Moreover, the plants showing severe symptoms of the disease had higher expression of
eIF4E gene. Hence, in the present study, up regulation of eIF4E gene in relation to BBrMV
infection was observed which indicates its role in disease development.
Plant Biotechnology
660.6 / GER/CH PG
Characterization of eIF4E gene in banana (Musa spp.) and analysis of its expression in relation to banana bract mosaic virus infection - Vellanikkara Department of Plant Biotechnology, College of Agriculture 2022 - 53p.
MSc
Banana and plantain (Musa spp.) are one of the most important food crops especially
in the tropics. India is the leading producer of banana in the world with a share of around 25
per cent in total output. Despite its importance, banana cultivation in India is impeded by
disease incidence and pest attack resulting in serious loss to farmers. Banana bract mosaic
disease caused by banana bract mosaic virus (family Potyviridae) is a major virus disease
affecting banana. Plant viruses need host factors for maintaining their life cycle. Eukaryotic
translation initiation factors, particularly the eukaryotic translation initiation factor 4E
(eIF4E) and its isoform are host factors essential for infection by plant viruses in the genus
Potyvirus. In many crop species, natural variation in eIF4E protein confers resistance to
potyviruses while in others, which lack natural eIF4E alleles conferring resistance, gene
editing can be applied to transfer genetic resistance. Therefore, gene editing approaches for
mutation of eIF4E gene have potential in conferring potyvirus resistance to plants. Sequence
characterization of eIF4E gene in Musa spp. is necessary for future studies on gene editing
for resistance breeding. Moreover, the expression studies of eIF4E gene in relation to
BBrMV infection will provide an insight into its probable role in disease development. In this
background, the present study entitled “Characterization of eIF4E gene in banana (Musa
spp.) and analysis of its expression in relation to banana bract mosaic virus infection” was
undertaken during the period 2019 to 2022 at the Department of Plant Biotechnology,
College of Agriculture, Vellanikkara, Thrissur, with an objective to characterize the eIF4E
gene in banana cultivar Nendran and to analyse its expression in relation to BBrMV
infection.
Initially, leaf samples were collected from Nendran banana plants from field at
Banana Research Station, Kannara, Kerala. Purification of total RNA was attempted using
TRI reagent (Sigma), Purelink Plant RNA reagent (Invitrogen) and RNeasy plant mini kit
(Qiagen). Out of the three methods, good quality RNA was obtained using RNeasy plant mini
kit (Qiagen). The total RNA was converted to cDNA using Oligo(dT)18 primer and reverse
transcriptase enzyme. This was followed by PCR amplification of the eIF4E gene using two
pairs of primers targeting overlapping regions of the gene. The PCR amplicons were
sequenced and the overlapping sequences were aligned to obtain the final contig of 853 bp
using Cap3 software. The BLASTN analysis revealed that the sequence showed 99.06 %
identity with 99 % query coverage with predicted sequence of Musa acuminata subsp.
malaccensis eukaryotic translation initiation factor 4E-1 (LOC103981303) (Accession no.
XM_009397982.2). The BLASTX search against NCBI protein database revealed that the
sequence showed 99.58 % identity with predicted protein sequence of eIF4E-1 in Musa
acuminata subsp. malaccensis (Accession no. XP_009396257.1). Five exons were identified
in the coding sequence. The amino acid sequence of the eIF4E gene was deduced from the
nucleotide sequence using ExPASy translate tool. Eight open reading frames were identified
using ORF finder. The multiple sequence alignment and phylogenetic analysis showed that
the Nendran eIF4E gene was more related to sequence of eIF4E gene of Musa acuminata
subsp. malaccensis.
Quantitative real-time PCR analysis was performed to study the differential
expression of eIF4E gene in banana cultivar Nendran under healthy and diseased conditions.
Five banana plants showing typical symptoms of BBrMV and a symptomless banana plant
were initially identified from the field and later confirmed for the presence and absence of
virus respectively based on diagnostic RT-PCR with BBrMV coat protein gene specific
primers. For gene expression study, two samples each were collected from the identified
diseased and healthy plants. One pair of primer each for banana eIF4E gene (test gene) and
actin gene (endogenous control) was designed using Primer3 software for the qRT-PCR
study. The real time PCR reaction based on SYBR Green chemistry was carried out for
quantification of expression of eIF4E gene in diseased and healthy Nendran banana. The
relative expression levels of the gene was normalised with the expression of endogenous
control actin gene following 2-∆∆Ct method. The diseased banana plants showed 1.48 to 16.92
fold increase in the expression of eIF4E gene when compared to the healthy plants.
Moreover, the plants showing severe symptoms of the disease had higher expression of
eIF4E gene. Hence, in the present study, up regulation of eIF4E gene in relation to BBrMV
infection was observed which indicates its role in disease development.
Plant Biotechnology
660.6 / GER/CH PG