1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Development of recombinant coat protein for immunodiagnosis of banana bunchy top and bract mosaic diseases(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Darsana Dilip, K C; Vimi LouisThe present investigation was undertaken to develop recombinant coat protein (rCP) of Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) for immunodetection of the viruses. The experiments were conducted at the Virology Lab, Banana Research Station, Kannara; Department of Plant Pathology, College of Agriculture, Vellanikkara, Kerala Agricultural University and Indian Institute of Science, Bengaluru during the period of 2016-2020. A roving survey in 10 districts of Kerala, divided into population subsets viz., North, Central and Southern zones were conducted for sample collection. After a preliminary DAC-ELISA, 17 and 12 representative samples respectively were selected and carried forward for further evaluations. The CP gene of BBTV was amplified from the total DNA isolated using reported primers by Polymerase Chain Reaction (PCR) and that of BBrMV by Reverse Transcriptase-PCR (RT-PCR). The CP gene sequences of these isolates were determined and submitted in the NCBI-GenBank Database. The 17 BBTV isolates were designated as MT174314-MT174330 and the 12 BBrMV isolates as MT818176- MT818187. It was inevitable to evaluate the molecular diversity of the viruses prior to devising nucleic- acid based and serological detection methods. The phylogeographic analysis depicted a clear demarcation of BBTV Kerala isolates based on geography whereas no such clustering was observed in the case of BBrMV isolates. Being an RNA virus, the molecular diversity of BBrMV (ranging between 1-12 %) was higher than BBTV. However, the 5’ and 3’ terminal of BBrMV CP gene was hypervariable and found unsuitable to be targeted for nucleic-acid based detection. Hence, forward primer was designed from the NIb region of ssRNA genome of BBrMV and reverse primer from 3’ UTR region upstream and downstream to the CP gene respectively. For nucleic-acid based detection of BBTV, highly conserved non-coding region of DNA-S upstream and downstream to the CP ORF was targeted. The primers were validated by detecting virus from the field samples collected from various parts of the state. The rCPs were chosen as a potential antigen for raising antibodies in order to develop serodiagnostic assays for the early detection of the viruses. The BBTV CP gene was clonedin to three expression vectors viz., pRSET-C, pGEX-4T-2 and pET32a(+) and transformed to expression hosts like BL21 (DE3) pLysS, Rosetta (DE3) pLysS and C41 strains of E. coli after amplification in DH5α. The 20 kDa recombinant BBTV CP (rBBTV CP) cloned in to pRSET-C, and overexpressed in various E. coli hosts had a hexa histidine (6X His) tag at the N terminal. Similarly, a 37 kDa fusion protein (pET/rBBTV CP) was overexpressed from pET/BBTVCP clone had a thioredoxin (Trx) tag (17 kDa) along with the 6X His tag. Whereas, a 45 kDa fusion protein (pGEX/rBBTV CP) with GST tag was overexpressed from pGEX/BBTVCP clone. These affinity tags in the fusion rCP enabled purification from other E. coli proteins. Although pRSET/rBBTV CP was soluble, the 20 kDa protein was highly unstable and partially degraded during purification at 4 °C. Curiously, pGEX/rBBTV CP dissociated from its GST affinity tag and the rCP without the tag degraded. On evaluating the protease cleavage sites in the fusion protein, trypsin cleavage sites were present between the C terminal of GST and N terminal of BBTV CP which might be the reason for cleavage of the ~20 kDa protein from its affinity tag. Thus, it was impossible to purify the protein from the pool of E. coli proteins. Restriction free (RF) cloning of BBTV CP to pGEX-4T-2 was attempted not only to replace these trypsin cleavage sites but also the thrombin cleavage site present in the vector with Tobacco etch virus (TEV) NIa protease site. Thrombin is a specific enzyme used to cleave off the tag from the fusion protein after purification. However, its specificity is not universal. Furthermore, the commercially available enzyme is costly. TEV protease on other hand was produced in the laboratory and was highly specific. However, the cleavage using TEV protease was unsuccessful apparently because of a steric hindrance contributed by the two extremely ordered regions flanking the TEV cleavage site present in the disordered region of the fusion protein. pET/rBBTV CP was highly soluble like ΔpGEX/rBBTVCP. Likewise, BBrMV CP gene was cloned into pRSET-C and pGEX-4T-2 to obtain pRSET/rBBrMV CP and pGEX/rBBrMV CP of size 34 kDa and 60 kDa respectively. The 34 kDa pRSET/rBBrMV CP was insoluble. Overexpression and purification of the protein was standardized in various conditions to increase solubility. On the contrary, pGEX/rBBrMV CP was highly soluble and was purified by GSH Sepharose affinity column chromatography. 360 μg/ml of untagged protein was obtained from 1 l culture. However, like any other potyviral CP, the exposed N and C terminal of BBrMV CP was also prone to proteolytic cleavage. It partially degraded when incubated with thrombin atroom temperature for GST tag cleavage. All these bands were detected by potyviral CP specific antibody in Western blot. Further on storage complete degradation of the protein was observed. Further standardisation of the protocol is necessary to either stabilise monomeric CP or develop BBrMV VLPs in vitro for immunising animal in order to raise the antiserum. The immunogenicity of the antigens (rBBTV CP and rBBrMV CP) was confirmed by Western blot using BBTV CP specific and potyvirus CP specific antibody procured from NRC, Banana and IISc, Bangalore respectively. The rCPs were also characterized by fluorescence spectroscopy, sucrose gradient ultra centrifugation and electron microscopy. The fluorescent spectra of tagged and tag less rBBrMV CP deviated from 330 nm which is typical for a partially disordered protein. However, the spectra of pET/rBBTV CP and ΔpGEX/rBBTV CP were different. The former depicted the spectra of a mostly globular protein. There were two λmax for the fluorescence spectra of ΔpGEX/rBBTV CP. The epitope prediction of BBTV CP with Trx tag gave interesting insights. A single linear epitope of 80 residues were detected in pET/rBBTV CP comprising of C terminal of the affinity tag and the N terminal of BBTV CP. This was expected to increase the immunogenicity of the antigen and administered for production of antiserum. The titre value of polyclonal antiserum produced against the 37 kDa pET/rBBTV CP was evaluated by DAC-ELISA and was found to be 1:128000. Titre value for serological assays of field samples was standardized as 1:10000 to be more inclusive for detecting virus even at early stages of infection. A total of 247 tissue culture samples and 10 field samples were screened for the presence of the virus using the antiserum and was compared with the procured antiserum. Seemingly, the latter non-specifically reacted with plant proteins which gave a higher absorbance value in negative control and correspondingly high absorbance in the infected samples. The polyclonal antiserum raised against rBBTV CP was used to standardize serological detection assays like IC-PCR, DIBA and TAS-ELISA apart from DAC-ELISA. DIBA and TAS-ELISA were the most sensitive assays which could detect up to 1:80 dilution of the antigen. In conclusion, due to the higher nucleotide variability of the CP gene, serological detection is preferred over nucleic acid based assays. However, the quality of antigen used for raising the antibody plays a major role in serodiagnostics. Hence, high quality rCPs of both BBTV and BBrMV were developed in the laboratory in various vector/host systems. ThepET/rBBTV CP overexpressed in C41 strain of E.coli (1.1 mg/ ml obtained from 1 L culture) was used for immunisation of the animal. A highly sensitive antiserum specific to BBTV with a titre ten fold higher than that of the commercially available antiserum was obtained. Using this antiserum raised against rBBTV CP, various serodiagnostic assays were standardised in the laboratory. Among these, TAS-ELISA was the most sensitive, detecting antigen even at higher dilution.Item 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 BThe 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.Item Computational prediction of mirnas in banana (musa spp.) and evaluation of their role in virus infection(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Kokila Sajeev, Anurag Mathew; Sony, K BThe study entitled "Computational prediction of miRNAs in banana (Musa spp.) and evaluation of their role in virus infection" was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Central Tuber Crops Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2016–2018. The objective of the study was to predict miRNAs in banana using bioinformatics tools and to validate and analyze their expression during BBrMV infection. Computational miRNA prediction tool NovoMIR was used for the prediction of miRNAs in banana genome. Analysis was performed with all the gene coding or nucleotide sequences of banana and 85 pre-miRNAs were predicted from 11 chromosomes. Most of the pre-miRNAs ranged from 140–180 nt. G+C% content of pre-miRNAs ranged from 24-77% and A+U% content ranged from 23-76%. MFE of pre-miRNAs were found by using an online application RNAfold web server. MFE of pre-miRNAs ranged from -20 Kcal/mol to -194.4 Kcal/mol. AMFE of pre-miRNAs ranged from -18.9 Kcal/mol to -85.4 Kcal/mol. All the predicted pre-miRNAs by NOVOMIR had MFE ≤ -20 Kcal/mol. MFEI of pre-miRNAs ranged from 0.85 to 1.82. BLAST analysis of 85 pre-miRNAs against annotated mature miRNAs in miRBase resulted in 52 mature miRNAs. The targets for the 52 mature miRNAs were predicted using the web tool psRNATarget and were functionally annotated by Blast2Go analysis server. Targets were identified for 40 miRNAs in banana genome. A total of 124 targets were found, with each miRNA having more than one target. Validation of the predicted miRNAs were done in in vitro banana plants of variety Nendran. Three months old tissue culture plants were infected with BBrMV virus by using aphids. Twenty healthy aphids were released on BBrMV infected sucker for 30 min for acquisition feeding, after starving for 10 min. These aphids were further released on tissue culture plants for 12-14 h for infection. The infection process was repeated twice a day for 7 days. For experimental validation, five miRNAs and their target genes having role in biological process were selected and stem-loop/gene specific primers were designed. RNA was isolated from healthy and infected leaf samples and reverse transcribed to cDNA. Expression analysis using RT-qPCR showed the presence of all the five miRNAs in healthy and BBrMV infected leaf samples. Out of them, miR-3900-5p, miR-9112 and miR-5417 were found up-regulated, miR-6928-5p downregulated and miR-3900-5p remain unchanged in infected samples and there was a positive correlation with the expression of their corresponding target genes. In the present study, 52 mature miRNAs have been predicted using bioinformatics tools in banana genome. Targets for 40 miRNAs were identified in banana genome. The five miRNAs selected were validated along with their targets. Expression analysis showed regulation of four of them during virus infection, indicating the possibility of their role in stress response in banana. The remaining miRNAs predicted need validation.Item Molecular cloning and characterisation of coat protein gene of banana bract mosaic virus(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Darshan Gowda, M R; Anita Cherian, KBanana (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.