A Preliminary Study on the Isolation and Construction of Partial cDNA Libraries of the Banana Bract Mosaic Virus (BBMV) Viral RNA

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.