Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Indeterminate growth of the umbel inflorescence and bulb is associated with increased expression of the TFL1 homologue, AcTFL1, in onion.

TERMINAL FLOWER1 (TFL1) is a key gene for maintenance of vegetative and inflorescence indeterminacy and architecture. In onion, flowering and bulbing are two distinct developmental phases, each under complex environmental regulatory control. We have identified two CEN/TFL1-like genes from onion designated as AcTFL1 and AcCEN1. AcTFL1 is expressed during bulbing and inflorescence development with expression increasing with indeterminate growth of the umbel and the bulb suggesting possible conservation of function. Increase in AcTFL1 expression during umbel growth is associated with a simultaneous reduction in expression of AcLFY. Expression of AcTFL1 within the bulb is lowest in the outermost layers and highest in the innermost (youngest) layers. Bulb storage at room temperature or in cold leads to a gradual reduction in AcTFL1 levels in the meristem-containing tissues, the decrease being faster in the variety not requiring vernalization. Constitutive expression of AcTFL1, but not AcCEN1 complements the Arabidopsis tfl1-14 mutant and delays flowering in wild type suggesting conservation of the AcTFL1 function even in the distantly related Arabidopsis. Taken together, AcTFL1 appears to be the functional counterpart of TFL1 and regulates indeterminate growth of the umbel inflorescence as well as bulb development in onion.

Analysis of synonymous codon usage bias and phylogeny of coat protein gene in banana bract mosaic virus isolates.

The banana is one of the world's most important livelihood crops. Banana plants are principally infected by four virus species, Banana bunchy top virus (genus Babuvirus), Cucumber mosaic virus (genus Cucumovirus), Banana streak virus (genus Badnavirus) and Banana bract mosaic virus (genus Potyvirus). The objective of this study is to understand the codon usage pattern and phylogeny of coat protein gene in different banana bract mosaic virus (BBrMV) isolates. The BBrMV Coat Protein (CP) gene was amplified from BBrMV infected banana plant samples collected from different districts of Tamil Nadu and Karnataka, India. Six new BBrMV isolates were submitted to National Center for Biotechnology Information. Phylogenetic analysis and codon usage indices were studied along with other isolates of BBrMV. Phylogenetic analysis of CP genes shows that most of BBrMV isolates are closely related to each other except KF385484.1 and KF385478.1. Relative codon usage patterns among different BBrMV isolates were calculated by software CodonW version 1.4.2. In BBrMV, codons with A-ended or U ended are the most preferential except the Leu and Gln whose optimized codons are CAG and UUG ending by G. The codon usage patterns of BBrMV isolates are principally influenced by mutational bias; however, compositional constraints along with mutational bias also play a major role.