1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Elucidation of role of small RNA mediated gene regulation in secondary metabolite pathway of black pepper (Piper nigrum L.)(Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, 2024-07-19) Shruthy, N S; Asha, SThe study entitled “Elucidation of role of small RNA mediated gene regulation in the secondary metabolite pathway of black pepper (Piper nigrum L.)” was conducted in the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2023-2024. Piperine, an amide alkaloid that contributes the unique spicy flavor is the major economic secondary metabolite of black pepper which is renowned for its pharmacological activities and therapeutic efficacies. The biosynthetic pathway of various secondary metabolites was highly influenced by a regulatory factor, microRNA- ‘the master regulators of gene expression’. They are small, non-coding RNAs of typically 18-24 nucleotides and can be a useful tool in metabolic engineering. Therefore, a better understanding of the mode of action of miRNA is very essential to exploit them in improving the plant for enhanced secondary metabolite production. Hence, the present study focuses on ‘identification and characterization of small RNAs involved in piperine biosynthesis from black pepper (Piper nigrum L.) In this study, the microRNAs targeting the key genes involved in the piperine biosynthesis pathway were analyzed by combined in silico-experimental method. From the 19 million reads of small RNA transcriptome, 303 conserved families of MiRNA were reported in black pepper. Among these, potential candidates of small RNAs were predicted to target the key genes involved in piperine biosynthesis such as Piperine synthase, piperamide synthase, and Piperoyl CoA Ligase genes. From these candidates, we characterized conserved and novel microRNAs. Precursor miRNAs for each candidate were also predicted based on their distinctive features such as length, ability to form hairpin stem-loop structure, and minimal folding energy. Among the potential candidate miRNAs, ‘Pni_miR5654’ and novel miRNAs such as ‘Pni_miR19’ and Pni_miR33’ were predicted for experimental validation. The tissue-specific expression of these miRNAs and their cognate targets in different parts of the black pepper variety Panniyur-1 were evaluated. The result indicates that the immature spike has the highest expression of microRNAs compared to leaf and mature berries whereas the highest expression of cognate target genes was observed in mature berries compared to leaf and immature spikes, that is, the miRNA: target pairs were inversely correlated. Further to analyse the expression of miRNA: target pairs in bio-elicitated callus cultures, initially the callus initiated from berries were treated with the culture filtrate of Pirimorsphora indica and R/NA was isolated at 0, 24, and 48 hrs after treatment. The study sowed and upregulation in the expression of both the miRNAs and their cognate pairs in 24 hour treatment. In this present study, the microRNA candidates Pni_miR5654, Pni_miR19’ and ‘Pni_miR33’ were identifies to have gene regulatory role in piperine biosynthesis by targeting the key genes in the pathway. Although miRNA medicated regulation was detected in the spike and berry stages, the inverse correlation of miRNA: target pairs was not found in in vitro cultures of black pepper, indicating the cellular reprogramming of miRNAs during callogenesis. The data generated from this study could be helpful in modifying the target genes, constructing artificial miRNAs, and performing inhibition of miRNA in order to enhance the production of piperine in black pepper.Item RNA mediated resistance against banana bunchy top virus (BBTV) in banana(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2024-02-17) Aparna, K Gokul; Vimi LouisThe study entitled “RNA mediated resistance against Banana bunchy top virus (BBTV) in banana” was conducted at the Department of Plant Pathology, College of Agriculture, Vellanikkara and National Research Centre for Banana, Trichy during the period from 2018 to 2023. The study aimed at developing banana lines that have resistance against Banana bunchy top virus (BBTV) through RNAi, using the ligation independent cloning (LIC) method. The RNAi approach targeted the replicase gene of BBTV. The process began with the amplification of replicase gene fragment that contains the dicer substrate siRNA region. Gene specific primers with adaptor sequences were used to amplify the DNA fragment, creating the sense and antisense fragments of the RNAi construct. The LIC vector which specifically contained four adaptor sequences (LIC1 to LIC4) was linearized with SmaI followed by treatment with T4 DNA polymerase and dTTP. Simultaneously, the amplified gene fragments were treated with T4 DNA polymerase in the presence of dATP, facilitating the development of 5' extending single stranded tails. Subsequently, the T4 DNA polymerase treated vector and gene fragments with sticky ends were mixed and incubated. The splicing of the cohesive ends on the insert fragments and the vector resulted in the circularization of the vector. The prepared vector, housing the desired sense and antisense fragments was transformed into E. coli DH5α. All the transformed colonies, obtained on antibiotic selection medium underwent initial screening via colony PCR, demonstrating cent per cent transformation. Further confirmation involved plasmid isolation, verification of intron, restriction digestion and sequencing. The next phase was transforming Agrobacterium EHA105 strain with the prepared vector by modified freeze-thaw method. The success of transformation was confirmed by verifying the presence of sense and antisense fragments through colony PCR. For plant transformation, embryogenic calli of banana cv. Nendran were developed from the immature male inflorescence of 0.5 cm in size, by culturing on MS medium supplemented with different combinations of hormones viz., 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA) and picloram. The embryogenic calli development was observed after five months, with the highest per cent in MS medium with 2,4-D (2 mg l-1) and IAA (1 mg l-1). In order to minimize the time delay, the embryogenic cell suspension (ECS) obtained from NRCB, Trichy was used for plant transformation. The co-cultivation of Agrobacterium carrying the desired construct and ECS was done in the presence of acetosyringone for three days, in the dark at 24 to 25 ⁰C, followed by washing of excess Agrobacterium with cefotaxime. The co-cultivated embryogenic cells were transferred to a selection medium containing kanamycin (100 mg l-1). The transformed selected embryogenic cells were then transferred to the embryogenesis medium for 35 to 40 days, followed by culturing on embryo germination medium, under light. The germinated embryos were cultured in Petri plates for one and half months, followed by regular subculturing and maintenance in tissue culture bottles, till they attained the three to four leaf stage. The plants were rooted in a rooting medium for 30 to 40 days, and then acclimatized in a containment facility. The PCR assay with the primers for amplification of sense and antisense fragments confirmed the presence of RNAi construct in the acclimatized plants. These plants were then tested for BBTV resistance by aphid (Pentalonia nigronervosa) challenge inoculation. The transformed plants remained healthy, while the typical bunchy top symptoms appeared within 25 days on the non-transgenic control plants. However, it was observed that two out of the forty eight transgenic lines gradually developed yellowing symptoms, indicating varying levels of resistance among the transformation events. Therefore, further evaluation of these transgenic lines is crucial to better understand the resistance levels. The study has successfully demonstrated the efficacy of transgenic lines in conferring BBTV resistance through RNAi approach. By utilizing the ligation independent cloning, the hairpin construct preparation was done in more efficient manner. The transgenic lines developed in the study represent a promising tool, offering the scientific community a proactive defense against unmanageable BBTV epidemics in the absence of resistant lines.Item RNA mediated resistance to Yellow vein mosaic virus in okra(Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2021) Kelkar Vipul Ganesh; Deepu MathewOkra (Abelmoschus esculentus L. Moench, Malvaceae) is one of the leading vegetable crops in hot and humid tropics. Unfortunately, this climate is conducive for many of the pests and diseases. Okra is susceptible to viruses such as Yellow vein mosaic virus (YVMV) and Enation leaf curl virus (ELCV), belonging to the genus Begomovirus (family Geminiviridae). Because of the favourable conditions prevailing in the coastal region, the losses in Kerala state are 60-100%, depending upon the stage of plant growth and the severity of infection. RNAi is one of the promising molecular biology approach against the viral diseases. Keeping the above facts in view, the present study “RNA mediated resistance to Yellow vein mosaic virus in okra” was taken up at the Centre for Plant Biotechnology and Molecular Biology, CoA, Thrissur from September 2017 to May 2021. The high yielding and YVMV susceptible popular okra cv. Salkeerthi was selected for the development of resistance using RNAi mechanism. Total DNA was isolated from the YVMV infected plant and part of the βC1 gene (187 bp) of the virus was amplified using primers VβC1F and VβC1R. Sequence information of PCR product has revealed that the gene is 90-95% identical with the Indian isolates. The βC1 gene sequence was analysed using IDT software and 10 siRNAs were found at three different position (19-44, 34-59, 99-124 bp). Through Restriction Mapper, it was confirmed that the sequence selected for the preparation of sense and antisense strand, do not possess recognition sites for SmaI, HindIII and MauBI restriction enzymes which are present in the pRNAiLIC vector. The output of VSupPred revealed that the fragment does not contain any Viral Suppressor Regions (VSRs), with a high prediction score (0.625). The hairpin RNAi construct harbouring the region of βC1 gene of β satellite of Begomovirus of okra was generated using pRNAi-LIC (CD3-1285) vector. The SmaI digested plasmid produced three fragments, vector backbone (9842 bp), Pdk intron (1641 bp) and ccdB gene (614 bp) and the digested plasmid was treated with dTTP. Product-1 was PCR amplified (215 bp) with VLIC1 and VLIC2 primers, using the DNA from YVMV infected plant as template. Product-2 was PCR amplified (243 bp) with VLIC3 and VLIC4 primers using product-1 as template. Product-1 and product-2 were eluted from the gel and treated with dATP. The dATP treated PCR products and dTTP treated SmaI digested plasmid were mixed together and ligated by incubation at 65ºC for 5 min. followed by 22ºC for 15 min. Ligated product was successfully transformed in competent cells of E. coli (DH5α) and incubated on LB medium containing Kanamycin and Chloramphenicol. Colony PCR was performed, the transformation efficiency was found to be 80%. Plasmid was isolated from the positive DH5α colony and sequenced using the primers VLIC5 and VLIC6. The sequence data had shown that both sense and antisense strands are at right position and direction. Plasmid containing ihpRNA-βC1 cassette was successfully transformed into the competent cells of Agrobacterium (GV3101) and incubated on LB medium containing Kanamycin, Chloramphenicol and Rifampicin. Colony PCR was performed, the transformation efficiency was found to be 100%. Plasmid was isolated from the positive GV3101 colony and sequenced using the primers VLIC5 and VLIC6. Sequence data has further confirmed that both sense and antisense strands are at right position and direction. The ihpRNA-βC1 cassette was successfully transformed into okra cv. Salkeerthi using in planta method of Agrobacterium mediated transformation. The transformation efficiency observed was 11.42% and the transformation was confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5. cDNA was prepared from the total RNA isolated from transformed and control plants. siRNA synthesis was confirmed using the primers VLIC1 and VLIC5 (400bp) and Ubiquitin gene was confirmed using the primer UBQ7 (187 bp). Silencing potential of the RNA interference of βC1 gene and the development of resistance was evaluated by keeping the 15-day old transformed and control plants along with YVMV infected plants inside containment facility, with whiteflies released into insect cage for infection. All the control plants and one transgenic plant have shown the YVMV symptoms after 10 days. Three transgenic plants were healthy with no symptoms. The present investigation was successful in the development of YVMV resistant okra plants carrying ihpRNA-βC1 using pRNAi-LIC (CD3-1285) plasmid vector. The further evaluation is needed in the coming generations for the identification of stable transgenic lines.