School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemIdentification of long non-coding RNAs in plant reproductive development( 2023-07)Linear and circular long non-coding RNAs (lncRNAs, and circRNAs) are regulatory RNA molecules that do not encode proteins but play critical roles in biological processes in responses to internal and external factors. These non-coding RNAs can originate from various genomic regions, including exonic, intronic, or intergenic regions and function based on their specific nucleotide sequences and structures. This thesis aims to advance our understanding of circRNA and lncRNA expression patterns and functional roles in plants during flowering, employing RNA sequencing and plant transformation techniques, with a focus on soybean flowering and Brassica rapa pollen development. Pollen development is a crucial process that plays a pivotal role in fertility and subsequent seed production. In-depth RNA sequencing investigations were conducted to examine circRNA expression during five distinct stages of pollen development in B. rapa. A total 1180 circRNAs were identified. These circRNAs were generally small, ranging from 100 to 600 nucleotides in length, comprised of one to two exons, and displayed an uneven distribution across all chromosomes. Further analysis demonstrated differential and stage-specific expression patterns of circRNAs, primarily showing an upregulation trend during pollen development. Comparative analysis using bioinformatics tools revealed that the identified circRNAs in B. rapa exhibited approximately 35% sequence conservation with circRNAs identified in Arabidopsis thaliana, while only about 3% sequence conservation was observed with circRNAs from other plant species. Functionally, the identified circRNAs in B. rapa were found to regulate pollen development by participating in diverse biological processes, including protein biosynthesis, meiotic and meiosis cell division processes, DNA processing, enzymatic activities, and carbohydrate metabolism. Moreover, the investigation identified 88 circRNAs containing binding sites for microRNAs (miRNAs), indicating their potential role as miRNA sponges in post-transcriptional gene regulation. Specifically, the circRNAs expressed in B. rapa pollen exhibited binding elements for various flowering miRNAs, such as miR156, miR164, and miR172, suggesting their potential involvement in pollen developmental processes. To validate the presence of circRNAs, nine circRNAs were selected and confirmed through experimental procedures, including the verification of back-splicing junctions using divergent primers and Sanger sequencing experiments. Soybean (Glycine max) is considered the world's major source of vegetable oil and protein. The transition from vegetative growth to flower development in soybean is triggered by exposure to short-day photoperiod, as it is a short-day plant. During floral transition in soybean, RNA sequencing data was generated from Shoot Apical Meristem (SAM) samples dissected from plants subjected to short-day treatment at four time points: 0, 2, 4, and 6 days after treatment. The expression profiling of circRNAs identified 384 circRNAs in soybean SAM, which were found to be predominantly short in length (300-600 nucleotides) and composed of two to four exons. Furthermore, an uneven distribution across the 20 chromosomes of soybean was observed. CircRNAs exhibiting short-day treatment-specific expression patterns were noted during the floral transition processes in soybean, with a greater number of circRNAs displaying upregulation following six days of short-day treatment. An analysis of circRNAs expressed in soybean SAM revealed that they possess longer flanking introns, which were significantly enriched with reverse complementary elements, potentially facilitating circRNA biogenesis. Additionally, circRNAs derived from genes involved in flowering-related hormones such as abscisic acid and auxin were also identified. The involvement of circRNAs in diverse biological processes, including SAM development, adaxial/abaxial axis specification, reproductive shoot system development, and the regulation of flower development, was unveiled through in-silico functional analysis. Furthermore, miRNA binding sites were found in 38 circRNAs, including those associated with development and flowering, such as miR156 and miR172, suggesting their potential function in circRNA-miRNA-mRNA networks that regulate gene expression during floral transition. The backsplicing of 26 selected circRNAs was validated through divergent primer amplifications or Sanger sequencing. The intricate gene regulatory network underlying the floral transition in soybean was illuminated by these findings, which highlighted the unique characteristics and potential functions of circRNAs during this process. To explore the role of lncRNAs in soybean flowering, a specific flowering-associated lncRNA known as NC_GMAXST00046315 was introduced into soybean via Agrobacterium-mediated transformation. PCR screening of the obtained transgenic lines was performed using specific primers for the reporter gene beta-glucuronidase (GUS), which successfully confirmed the presence of the transgene in the transgenic lines. The quantitative PCR (qPCR) analysis revealed that the inserted lncRNA in the soybean genome exists in a copy number ranging from one to three copies. Moreover, qPCR analysis validated successful overexpression in selected transgenic lines, with expression levels ranging from 1.8- to 2.35-fold higher compared to non-transgenic plants. Since lncRNAs typically regulate the expression of their neighbouring genes in cis or trans, further investigations were undertaken to assess whether the expression of neighbouring genes was influenced by overexpression of the lncRNA. Specifically, the impact of lncRNA overexpression was compared between non-stress and heat stress conditions, as the BAG gene (heat-responsive gene) is located downstream of the lncRNA. The results indicated a significant decrease in the expression of BAG in transgenic lines compared to WT plants (non-transgenic Bragg) under non-stress condition. During heat stress, a noteworthy increase in lncRNA expression was observed in WT plants, implying its involvement in stress-responsive pathways in soybean. The expression of the BAG gene exhibited a 270-fold increase in WT plants and up to 680-fold increase in transgenic lines, indicating the potential regulatory effect of the overexpressed lncRNA on BAG gene. The transgenic soybean lines also exhibited notable improvements in seed characteristics, resulting in significantly higher yields compared to WT plants. Specifically, the transgenic lines displayed enhanced seed weight and a higher percentage of undamaged seed coat, while WT plants produced smaller seeds with wrinkled discoloured seed coat. The seed weight of transgenic lines increased by approximately 15%, and the occurrence of intact seed coat increased by 6-9% compared to WT plants. These findings highlight the positive impact of lncRNA overexpression on soybean yield, particularly under heat stress conditions. In summary, the significance of circRNAs and lncRNAs in plant flowering and reproduction is emphasized by this research. The identification of critical regulatory circRNAs and lncRNAs in this study contributes to a deeper understanding of the regulatory mechanisms that control flower development in crop plants. Furthermore, this knowledge has the potential to facilitate the advancement of more efficient and higher yielding seed-producing crops.