PG Thesis

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    Identification and characterisation of virus responsive miRNAs in banana musa (AAB) Nendran
    (Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Athira Subramanian; Soni, K B
    The study entitled “Identification and characterization of virus responsive miRNAs in banana Musa (AAB) ‘Nendran’” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to identify the miRNAs associated with Banana Bract Mosaic Virus (BBrMV) infection in banana var. Nendran from the expression profile of selected miRNAs. Five miRNAs were selected from the 52 computationally predicted miRNAs in a previous study conducted in the Department of Plant Biotechnology. Selection was based on the function of their target genes i.e. their role in biotic stress conditions. The miRNAs selected were miR-3900-5p (targets: Plant viral response family protein and Putative disease resistance protein genes), miR-2172-5p (target: Putative ethylene responsive transcription factor1gene), miR-6928-5p (target: Flavin adenine dinucleotide dependent oxidoreductase gene), miR-5417 (target: Stress endoplasmic reticulum protein 2 gene) and miR-971-5p (targets: Argonaute protein and Transport inhibitor response-1 like protein genes). For studying the expression of miRNAs, three month old in vitro raised banana var. Nendran plants were infected with BBrMV through viruliferous aphids (Pentalonia nigronervosa) by acquisition feeding method. Fifteen to twenty aphids reared in healthy banana suckers were transferred to the BBrMV infected sucker for 2 hour acquisition feeding, after starving for 30 min. These aphids were released on to the leaf axils of the tissue culture plants for a period of 24 h. After this time period the aphids were killed using an insecticide. Infection was confirmed by PCR using replicase specific primers and the results showed the presence of BBrMV specific amplicons from 24h onwards in all the samples. For studying the expression of these miRNAs, leaf samples were collected from the infected plants 24 h, 48 h, 1 wk and 2 wk after infection. RNA was isolated using CTAB method, reverse transcribed to cDNA and PCR was done. PCR analysis confirmed the presence of all the five computationally predicted miRNAs in banana. The expression profiles of the miRNAs and their target genes were studied by RT-qPCR. The results showed upregulation of miR-3900-5p, miR-2172-5p, miR-6928-5p and miR-971-5p (1.2, 2.0, 1.27, 2.0 fold respectively) 24h after BBrMV infection. Among them miR-3900-5p, miR-2172-5p and miR-971-5p maintained higher expression upto one week compared to uninfected control. miR-6928-5p showed a 1.72 fold increase in expression 48 h after infection. Expression of miR-5417 was down regulated by BBrMV infection at 24 h of infection. The two targets of miR-3900-5p (Plant viral response family protein and Putative disease resistance protein genes) showed contrasting trends in their expression after BBrMV infection. While Putative disease resistance protein showed a drastic increase (13 fold) 24 h after infection, Plant viral response family protein expression showed a continuous reduction throughout the period of observation. miR-2172- 5p and its target Putative Ethylene-responsive transcription factor 1gene (3.39 fold) were found upregulated during the infection. While miR-6928-5p showed maximum expression at 48 h, its target FAD (Flavin adenine dinucleotide) dependent oxidoreductase gene showed maximum expression at 24 h after infection and maintained a higher level upto 48 h. Among the two targets of miR-971-5p, Transport inhibitor response-1like protein gene showed a quick response to virus infection with a 6.15 fold expression at 24 h, while Argonaute protein gene showed a lower expression upto 48h and a drastic increase upto 2 fold as the infection progressed. The study suggested that miR3900-5p, miR2172-5p, miR6928-5p and miR971-5p were associated with BBrMV infection in banana var. Nendran. Their targets viz., putative disease resistance protein gene, putative ethylene responsive transcription factor 1gene, FAD dependent oxidoreductase gene and transport inhibitor response-1 like protein gene showed significant increase immediately after the infection. Suppression of the targets viz., Plant viral response family protein and Argonaute protein genes during BBrMV infection suggested the possible role of miR-3900-5p and miR-971-5p in regulating the infection process.
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    Development of a nano biosensor for detection of bract mosaic virus in banana (Musa spp.)
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Saurav Saha; Abida, P S
    Banana bract mosaic virus (BBrMV) is a recently described virus of banana which contributed to yield reduction by 5 to 36 per cent and is a barrier to international exchange of germplasm. There is a no effective measure to control this virus, only by routine virus indexing of planting material can protect the spreading of this virus. Currently ELISA and real time PCR is effectively used for diagnosis but the protocol is time consuming and expensive. In the recent years biosensor based on the novel metallic nanoparticle gain much importance for industrial applications and efficient detection of viruses. The study entitled ―Development of a nano biosensor for detection of Banana bract mosaic virus in banana (Musa spp.)‖ was carried out in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture and Centre for Electronics and Materials(C-MET), Thrissur during the academic year 2014-2016. The objective of this study to develop an antibody based nanobiosensor for quick detection of Banana bract mosaic virus. Goldnanorods (GNRs) were fabricated through seed-mediated procedure and UV-Vis spectra of GNRs solution indicated characteristic longitudinal and transverse band at 710 nm and 520 nm respectively. The transmission image of electron microscope revealed that solution contain rod shaped gold nanoparticles with length and diameter (42±3) nm (14±1.9) nm respectively. The aspect ratio of GNRs was measured through ImageJ software and found that aspect ratio of GNRs was 3.03. The effect of silver nitrate solution on the growth of GNRs was studied and found that with increasing silver ion concentration in a growth solution longitudinal peak shift was observed from 710-740 nm and also aspect ratio of GNRs also increased from 3.03 to 3.75. In order to detection of analyte (BBrMV) surface of a GNRs activated with complete replacement with alkalithiol molecule for covalent attachment of an antibody. UV-Vis spectra of activated GNRs indicated that due to formation of SAM (Self assembly monolayer)position of a peak shifted from 710- 716 and also due to binding of an antibody to SAM layer again peak position was changed from 716-727nm. SDS-PAGE and Nanodrop spectrophotometer analysis were carried out for the BBrMV antigen to check the quality and quantity of protein (antigen). The results had shown 38KDa band coat protein specific band of virus in a gel and concentration of antigen was 3mg/ml. Bio-recognition induced gold nanorods aggregation here takes as an analytical tool for detection of a BBrMV. In this case due to addition of antigen to antibody labeled GNRs solution. Colour of the solution changed red to black and notable peak shift of (7- 25) nm was observed both in transverse and longitudinal peak of GNRs in UV-Vis Spectra. Antigen concentration up to 0.25 mg/ml and above shows stability in the Peak shift and colour change in infected sample compared to control sample. In healthy sample no colour changes were observed and with only minimum peak shift was there. The positive result was also obtained in a micro titre plate where ELISA reader clearly differentiated healthy and infected samples with different concentrations of antigen. In case of longitudinal peak shift, the kinetics curve of an infected sample remained relatively flat and after 15min the shift remained stable until the end of the observation and the absorbance of GNRs continuously decreased up to 80th min and after that no changes were observed in the kinetics curves of an absorbance. For determining the accuracy and sensitiveness of a nanobiosensor, results of the different serological techniques (ELISA, DIBA) were compared with the result of the fabricated solution based nanobiosensor and found that nanobiosensor could detect the viral protein at a very low concentration (2-0.02) mg/ml, whereas in the case of other techniques the detection was possible up to 0.12 mg/ml of antigen concentration. The developed gold nano rods based nanobiosensor was evaluated for detection of a BBrMV in five varieties of banana and it was found that cv. Nendran was more affected by BBrMV compared to other varieties of banana due to the high concentration of viral load in the infected samples. The solution based gold nano rod based biosensor is sensitive, cost effective and easy for virus indexing of tissue culture plants and planting materials compared to other methods currently in use. Further investigations and refinement could lead to the fabrication and development of nanobiosensor on a commercial scale.