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Molecular Characterization and Expression Profiling of Genes Involved in Ripening Banana

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Title Molecular Characterization and
Expression Profiling of Genes Involved in Ripening Banana
 
Creator Lokesh, V.
 
Subject 03 Biochemistry & Molecular Biology
02 Banana
 
Description Though phytohormones and signaling compounds are known to play
important roles in ripening, exact mechanisms through which these molecules are
involved in ripening physiology are not completely known except for the involvement
of the phytohormone - ethylene. As climacteric ripening is associated with hundreds
of genes that are regulated by many factors, differential display RTPCR (DDRTPCR)
coupled with silver staining was performed to identify some of the differentially
regulated genes by the phytohormones (ethylene, gibberellic acid, indole-acetic acid,
abscisic acid and kinetin) and signaling compounds (salicylic acid and nitric oxide).
Using 10 primer combinations and 40 populations of mRNA (5 stages of each
treatments including control), a total of 68 bands were obtained, of which 52 were
cloned into T/A cloning vector and sequenced. A total of 32 differentially regulated
genes were identified after removing all the redundant and false positive sequences.
These 32 sequences were identified, based on homology search using NCBI-BLAST
as well as BLAST search in banana genome database. All the sequences were
submitted to NCBI-EST library and the accession numbers were obtained. The
sequences were categorized into groups based on their putative functions, such as in
stress and senescence, in transcription and translation related, transport, carbohydrate
metabolism and some with unknown functionalities. In addition to these reported
proteins, several unknown gene fragments were also identified for the first time in
ripening fruits of NR. The differential expression of these genes was further
confirmed by quantitative real time PCR to know the exact extent of change in
expression level. The identified differentially expressed genes could be further
explored for their actual roles in ripening in relation to phytohormones and signaling
compounds. The information generated through experiments of this chapter are useful
in elucidating the role of the above- mentioned phytohormones and signaling
compounds in ripening.
The identification of full length gene sequence especially the coding sequence
(CDS) is a crucial step in the functional annotation of genes. CDS is a sequence of
nucleotides that corresponds to a specific sequence of amino acids in a protein. Apart
from CDS, the other non-coding UTR regions at 5’ and 3’ end of the have also been
of great significance owing to their significance in the regulation of the corresponding
Abstract
gene expression. From the 32 genes that were differentially regulated under the
influence of various phytohormones and signaling compounds, three genes viz., 1-
aminocyclopropane carboxylate oxidase (ACO), probable galacturonosyltransferase 7
(GAUT-7) and ubiquitin which were of great relevance either to fruit ripening or to
phytohormone signaling were amplified for full length gene sequence using RACE
approach. ACO full length gene sequence was 1159 with 957 bp CDS. GAUT-7 full
length gene sequence was 2156 bp with 1359 bp CDS and the other full length gene
coding for ubiquitin was 448 bp and the ORF region was of 234 bp. All the genes
were analyzed for their transcription start site, miRNA target sites at 3’ UTR using
several online tools. The gene sequences were also checked for their similarity with
the corresponding AAA group banana gene sequences and were highly similar to the
AAA group gene sequences.
The prime task of shelf life extension for tropical climacteric fruits such as
banana relies on the suppression of ethylene through controlled elicitation of certain
metabolic regulators, which in turn counter ethylene formation. NO’s role in
delaying/ inhibiting fruit ripening has been of great interest and many delivery
systems are being practiced. Various NO donors were screened for their efficacies in
releasing NO in a desirable manner for enhancing fruit attributes. Among several NO
donors, SNP at 1 mM concentration appeared beneficial and hence was further
analyzed for its effects on the nutritional profile of banana fruits. While the nutrition
profiles were not affected, the SNP treatment enhanced fruit quality by enhancing
shelflife and inhibiting phenylpropanoid pathway enzymes like PAL and PPO.
Among many proposed mechanisms of NO regulation in plants, information on
polyamine (PA)-mediated regulation is poor. Owing to both NO and PA’s role in
fruit ripening control, the effect of NO in altering PAs level and the resulting
modulation in ethylene level was analyzed by studying the alterations in PA levels
after SNP treatment.. It was found that putrescine content increased by 5.8 and 9
fold in pulp and peel respectively. Spermine was induced more in peel (10.2 folds).
Spermidine levels were induced by 2.9 and 8.3 folds in pulp and peel of Cavendish
banana respectively. The expression studies of ethylene pathway genes ACS and
ACO showed that they were downregulated in both Cavendish and NR fruits. ACS
was downregulated by 5.2 and 7.2 folds on the 5th day. The other ethylene pathway
gene ACO was also downregulated by 3.1 and 4.5 folds in Cavendish and NR banana
Abstract
fruits respectively. PA pathway genes were found upregulated in at least few stages
of ripening after the SNP treatment. ODC was found upregulated by 3.8 and 2.3 folds
in Cavendish and NR banana fruits respectively. SAMdC was upregulated by around
8 fold in Cavendish and SPMSYN and SPDSYN were also found consistently
upregulated by SNP treatment. However, ADC was the gene which recorded the
maximum upregulation by 14.93 and 10.4 folds in Cavendish and NR banana fruits
respectively thus suggesting induction in putrescine levels by NO occurs via
agmatine by up-regulation of ADC and probably not much via ornithine and ODC.
Effect of various phytohormones and signaling compounds (Gibberellic acid,
Auxin, Cytokinin, Abscisic acid, Salicylic acid, Methyl jasmonate, and Nitric oxide)
for inhibiting/ delaying ripening was evaluated. Expression pattern of genes involved
in diverse class of biochemical and physiological functions like cell wall hydrolysis
(PG, PL, PME, β-galacturonase, Exp1 and Exp2), carbohydrate metabolism (Sucrose
synthase, SPS, α-amylase and β-amylase), ethylene pathway (ACS and ACO),
ethylene signal perception and transduction (ERS1, ERS2, EIL1, EIL2, EIL3, EIL4,
MADS1, MADS2, MADS4 and MADS6), senescence and defense (CAT, SOD, PAL
and chitinase) was studied. Though the maximum inhibition of ripening was imparted
by gibberellic acid, it could not down-regulate all class of genes. Diverse effect was
imparted by different growth regulators on various classes of ripening-related genes.
Based on genetic expression data, various formulations of these phytohormones and
signaling compounds were developed and were analyzed for their efficiency to delay
fruit ripening.
 
Contributor Bhagyalakshmi, N.
 
Date 2015
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/12168/1/Lokesh%20-%20Ph.D.%20thesis%20final%20-%2005.08.2015.pdf
Lokesh, V. (2015) Molecular Characterization and Expression Profiling of Genes Involved in Ripening Banana. PhD thesis, University of Mysore.