Molecular cloning and characterisation of coat protein gene of banana bract mosaic virus

Abstract

Banana (Musa spp.), identified as ‘tropical treasure’ is grown extensively in the tropical and sub tropical regions of the world. Diseases, especially those caused by viruses are major constraints for the profitable cultivation of banana. Among the viral diseases, banana bract mosaic is one of the most important, which leads to an yield reduction ranging from 52 to 75 per cent. This disease is caused by Banana bract mosaic virus (BBrMV) which is a member of genus Potyvirus and family Potyviridae. In case of any viral disease, early diagnosis is very important since symptomless hosts carry the viral inoculum. Development of molecular clones of viral genome has immense application in the field of disease diagnostics and management. Hence, the present study was carried out with the objective to develop molecular clones of coat protein (CP) gene of BBrMV and to characterize it.The infected suckers were collected from Banana Research Station (BRS), Kannara and maintained in the insect proof net house of Department of Plant Pathology. The symptoms developed on different plant parts under natural field conditions were documented which included longitudinal, irregular, reddish streaks of varying sizes on the base of pseudostem, mosaic pattern on bracts, fan like orientation of leaves, spindle shaped lesions on leaves, reduced bunch size and malformed fingers.The serodiagnostic technique namely, Direct Antigen Coating-Enzyme linked immuno sorbent assay (DAC-ELISA) was validated by determining the antibody titre with different dilutions of primary antibody viz., 1:100, 1:200, 1:300, 1:500 and it was found that BBrMV could be best detected at 1:200 dilution along with 1:500 dilution of secondary antibody. Later, the presence of virus particles in the samples were confirmed by DAC-ELISA using the standardized combination of primary and secondary antibody dilutions. Dot Immuno Binding Assay (DIBA) was validated to detect BBrMV and showed positive reaction for infected leaf sample which was detected by the development of purple coloured spots on nitrocellulose membrane.The genome of BBrMV is RNA and hence, molecular detection of virus was standardized by Reverse Transcription- PCR (RT-PCR). Total RNA was isolated by two different protocols using different reagents. Among the two methods, the one with Ambion Purelink RNA Reagent was the most appropriate for RNA isolation from banana since it provided highest quality and quantity of RNA compared to the protocol with TRIzol reagent. The isolated RNA was converted into complementary DNA (cDNA) using First Strand cDNA synthesis kit. RTPCR amplification of coat protein gene was standardized using gene specific reported primer (B1/B2) and designed primer (BCPF1/R1) which yielded amplicons of approximate size, 605 bp and 850 bp respectively. The molecular cloning of CP gene was done in Escherichia coli (DH5- alpha). The presence of gene insert in transformed colonies were confirmed by colony PCR using plasmid specific primer (T7 and SP6) which yielded amplicons of expected band size of 1150 bp. The amplified colony PCR products were sequenced to obtain CP gene sequence of BBrMV. The characterization of cloned CP gene of BBrMV was carried out by in silico analysis. The blast analysis revealed that the CP gene sequence of the virus showed maximum homology of 99 per cent to KER2 isolate from Kasargod, Kerala (Accession no. KF385491). The sequence exhibited significant nucleotide identity (99 to 96 per cent) and amino acid identity (95 to 83 per cent) with other nucleotide and protein sequences of BBrMV available in the database of Genbank. The phylogenetic analysis by the alignment of CP gene sequences of selected 22 isolates also revealed that the present isolate was more similar to KER2 isolate and the Indian isolates did not show any relationship based on geographical origin.The recombinant clones developed in the present study could be applied in serodiagnosis and genetic engineering. This could be also used as disease diagnostic probes for more sensitive molecular diagnostic techniques like Nucleic acid spot hybridization.