Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)
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Date
2013
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
Banana is one of the important fruit crops of India. Banana is susceptible
to several fungal pathogens, nematodes, viruses and insect pests. The greatest
threats to global banana production is Fusarium wilt or Panama wilt caused by
Fusarium oxysporum f. sp. cubense. Control of the pathogen is difficult and
mainly involves the use of disease free suckers. Although disease resistance exists
in some banana cultivars, introducing resistance into commercial cultivars by
conventional breeding is difficult due to its triploid nature and sterility factors of
banana.
The study entitled "Gene expression analysis in relation to Fusarium wilt
resistance in banana (Musa spp.)" was carried out at the Centre for Plant
Biotechnology and Molecular Biology, Vellanikkara during the period 2009-2013
with an objective to identify differentially expressed genes in disease resistant
genotype of banana, Palayankodan using the molecular technique called
suppression subtractive hybridization (SSH).
Total RNA and mRNA were isolated from healthy and inoculated plants
(with Fusarium oxysporum f.sp. cubense) and were used respectively as 'driver'
and 'tester' in SSH reaction. The reactions were performed utilizing the PCR
select" cDNA subtraction kit provided by CLONTECH, USA.
Control subtraction was carried out first using PCR select" cDNA
subtraction kit, which gave satisfactory and expected results. For experimental
subtraction, the double stranded cDNAs synthesized from Zug mRNA from
normal 'driver' and treated 'tester' were digested with RsaI enzyme. Two tester
populations were created and each ligated to two different adaptors. This was
followed by two hybridization reactions and finally a selective PCR amplification.
Only differentially expressed cDNAs were amplified exponentially. This was
confirmed by analyzing the PCR products on agarose gel, which showed a smear
ranging from 0.9 to 1.3 kb in the subtracted sample and was different from smear
pattern of unsubtracted ones. The cDNA fragments from subtracted sample were
cloned in pJET and pGEMT vectors and sequenced. Fifty clones were sequenced
and analysed after vector and adaptor editing.
In silica analysis using bioinformatics tools revealed that some of the
cloned sequences showed similarity with known sequences which play important
roles during disease resistance conditions directly or indirectly. These included
resistance gene candidate NBS type protein, mitogen activated protein kinase,
phytoene desaturase, glycerol 3-phosphate dehydrogenase, neutral invertase, 1-
aminocyclopropane-l-carboxylase synthase, superoxide dismutase, MADS-box
protein, ubiquitin 2, actin, NADPH oxidase, phytoene synthase, ACC synthase,
sucrose phosphate synthase, phosphatidic acid phosphatase-like protein, ORF III
like polyprotein, bHLH transcription factor like protein, cytochrome oxidase,
isochorismatase hydrolase, basic helix-loop-helix family protein, constitutive
triple response I-like protein, granule bound starch synthase, alpha amylase
precursor, rop protein, GTPase family protein, S-adenosyl-L-methionine synthase
protein, ADP-glucose pyrophosphorylase glucose-l-phosphate adenylyl trans,
ethylene signal transduction factor and ribosomal protein. Clones were classified
into 6 major groups based on function of protein. Sequences had conserved
domains for the above mentioned proteins. Genes involved in defense, signal
transduction, metabolism, hypothetical protein, transcription factor and
translation. For further exploitation of these sequences it is necessary to clone full
length cDNA. ESTs thus generated in the present study will be of great use in
future for further downstream applications.
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