Expression of Recombinant Protein of Coat Protein Gene of Sweet Potato Feathery Mottle Virus in Bacterial Expression System and Production of Polyclonal Antiserum for Its Diagnosis

Sweet potato (Ipomoea batatus L. (Lam), Family Convolvulaceae) is one of the most important tuber crops providing nutritional security because of its high consumption value and medicinal properties, and numerous agro-industrial uses. Sweet potato feathery mottle disease caused by Sweet potato feathery mottle virus (SPFMV) is one of the serious constrains in sweet potato cultivation in India. Effective diagnostic methods need to be developed to solve the problem due to these viruses. As part of the study, infected leaf samples from fields were collected, positive samples were screened for SPFMV using DAC-ELISA and confirmed through PCR. Coat protein gene of SPFMV was PCR amplified, cloned into TA cloning vector and then transformed into Escherichia coli DH5α cells. Positive clones were sub cloned into expression vector pET28A(+) and transformed into DH5α cells. Plasmid DNA from positive clones were isolated and transformed into BL21DE3 cells (NiCo21-DE3 cells). Positive clones were identified and confirmed in-frame position through sequence analysis. Selected colony was grown in Luria both medium at 37oC. Cells were collected and solubility of SPFMV coat protein (CP) was checked through SDS PAGE. Various standardisations were carried out for optimising expression of SPFMV CP and it was observed that 4 hr induction of 1.5 mM IPTG at 25oC gives maximum yield. Using these conditions, cells were grown on large scale and purified the protein (SPFMV CP) using Ni-NTA resin affinity chromatography. Purified protein was checked using SDS PAGE, confirmed the expression using Western Blotting and given for immunization into two New Zealand white rabbits for polyclonal antibody production. Serological tests like ELISA and DIBA were done for confirming the sensitivity and specificity of the raised antibody using field samples of SPFMV infected sweet potato along with healthy plants. Tested samples gave strong positive reactions at dilutions of 1:500 up to 1:6000. Also antibody reacted specifically at a dilution of 1:6000 in ELISA and DIBA. This is the first report of development of polyclonal antiserum against CP of SPFMV through recombinant technology in India and can be useful for the detection of virus from the field-grown samples.

Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica)

Abstract‘HoneySweet’ plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, ‘HoneySweet’ DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum ‘Improved French’. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the ‘HoneySweet’ transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.