Gene expression analysis in relation to fusarium wilt resistance in banana (Musa spp.)

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.