Abstract:
Banana bract mosaic virus (BBMV), the cause of banana
bract mosaic disease, causes up to 40% yield loss in banana
production in the Asia and the Pacific region. Information on the
genomic organization of the virus can help in viral disease
management. The research aimed to study the BBMV helpercomponent
protease (HC-pro) gene by isolating and constructing
partial complementary DNA (cDNA) libraries. Total RNA was
extracted from infected “Saba” banana tissues and agarose gel
electrophoresis yielded the approximate 10 kb BBMV RNA. Two
primer pairs, namely HC2 and T5, were designed to amplify partial
sequences of the HC-pro gene (435 and 629 bp respectively). A
multiplex RT-PCR using previously designed primers, Core F2
and Core Rv2 to amplify the 324 bp core region of the coat protein
gene of BBMV, and the TS primers was done as an optimization
experiment. The HC2 and T5 primer pairs were used in RT-PCR
separately and yielded their respective gene fragments. The
amplified cDNA libraries were ligated into pGEM®-T Easy vector
(Promega) at 4°C with a 3:1 insert to vector molar ratio. The
recombinant plasmid constructs, designated as pGBCR, pGBT5p,
pGBT5, and pGBHC2, were subsequently transformed in
competent Escherichia coli DH5a. The desired transformants were
selected based on color screening on LB/amp/IPTG/X-gal plates
and further analyzed by plasmid isolation and restriction enzyme
digestion. Isolated plasmids showed the expected 3.399 kb
pGBCR, 3.450 kb pGBHC2, and 3.644 kb pGBT5p/pGBTS.
Single digestion of pGBCR with Pst I, pGBT5p with Bgl J,
pGBHC2 and pGBTS5 with EcoR I further confirmed the presence
of their respective inserts after agarose gel electrophoresis. The
nucleotide sequences of the plasmids (pGBCR and pGBTSp) and
amplified cDNAs (HC2 and T5) were determined using an ABI
PRISM" 310 Genetic Analyzer.
