Department of Plant Pathology, Regional Agricultural Research Station, Acharya N.G.. Ranga Agricultural University, Tirupati-517502, India.
Among the diseases and pests that attack groundnut, peanut bud necrosis disease (PBND) is a serious limiting factor in productivity of groundnut and widespread in all places wherever it is grown. Causative agent of PBND is tripartite RNA virus known as Peanut bud necrosis virus which belongs genus Tospovirus of family Bunyaviridae. The PBND infected groundnut samples collected from Tirupati were being maintained on Cowpea (cv. C-152) by mechanical sap inoculations. Total RNA was extracted from symptomatic plants of cowpea using RNA easy isolation kit (Quiagen) and its purity and integrity was checked on denatured agarose electrophoresis using TBE buffer containing guanidine isothiocyanate (GITC). The PBND coat protein gene was obtained by RT-PCR amplification of total RNA using coat protein gene specific primers (PBND-F:5’ATGTCTAACGTYAAGCARCTC-3’and PBND-R: 5’-TTACAATTCCA GCGAAGGACC-3’). The 800bp PCR product was gel eluted, purified and cloned in pTZ57R vector (Fermentas) and sequenced (EF179100). The DNA sequencing showed that the cloned coat protein gene was 831 bp in size and codes for 276 amino acids. The sequence obtained was compared with other PBND coat protein gene sequences available in the gene bank. Sequence analysis revealed that the PBND-TPT isolate infecting groundnut has 99% homology at DNA and amino acid level to ICRISAT (PBU27809)and Gadag (AY512647) isolates. The coat protein genes of PBND infecting fieldbean and cowpea under natural conditions at Tirupati were also cloned and sequenced (EF179099, 532937). The coat protein gene was further sub-cloned into bacterial ex-pression vector pQE-30UA (Qiagen) and transformed in E.coli M15 strain. The transformed E.coli M15 cells were incubated for 4 hours in LB broth and protein ex-pression was induced by adding 0.1M IPTG. The expressed protein was further confirmed by Western blot. The results showed that the size of expressed PBND coat protein was 30 kD. The expressed protein is used for preparing polyclonal antiserum against PBND.
