In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural p...In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.展开更多
Plants face biotic and abiotic stresses during their entire life cycle, which leads to the loss in crop productivity. It has been shown that a relatively shorter exposure to heat stress, called priming, results in bet...Plants face biotic and abiotic stresses during their entire life cycle, which leads to the loss in crop productivity. It has been shown that a relatively shorter exposure to heat stress, called priming, results in better adaptation of plants under subsequent stresses, which plants may face. While rice plants in nature often encounter high temperature stress conditions, the strategies to cope with those are poorly understood. We identified the involvement of microRNA pathways in the adaptation to heat stress (HS) at the physiological and molecular levels. It was observed that osa-miR169 levels are altered after HS and in response to light conditions. Its expression was also regulated by heat priming during anthesis and effectively responds to the successive exposure to high temperature stress during grain filling in rice. Osa-miR169 targets nuclear factor Y (NF-Y). We propose that osa-miR169: NF-Y regulatory module may be important for HS memory induced during high temperature priming and thus may serve to integrate stress responses with light regulated development. The future study in this direction will be useful to understand how plants acclimatize to the changing environment and thus help in generating stress tolerant crops.展开更多
RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability ...RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability to restrict the RNAi machinery to ensure successful systemic invasion. The B2 protein of insect Flock House Virus (FHV-B2) and AC2 protein of Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are two well-characterized suppressors of RNAi, capable of reversing reporter gene silencing. In this study, we compared the strength of the two suppressors by assaying for the degree of RNAi reversion and the duration of sustaining the reversal in planta. The suppression activity was observed by assaying for GFP fluorescence at 3 dpi, 7 dpi and 14 dpi. The phenotypic observations were corroborated with small RNA Northern Blotting and semi-quantitative RT-PCR. The results indicate that suppressor strength of FHVB2 is comparable to MYMIV-AC2, although they are encoded by virus infecting host from two different eukaryotic kingdoms. This study will provide new insights to dissect the conservation in the RNAi pathways during the host-virus interactions.展开更多
microRNAs (miRNAs) are key regulatory molecules that fine tune gene expression at the transcriptional and post-transcriptional levels through the RNA silencing pathways. They play an important role in regulating plant...microRNAs (miRNAs) are key regulatory molecules that fine tune gene expression at the transcriptional and post-transcriptional levels through the RNA silencing pathways. They play an important role in regulating plant growth, development and response to a/biotic stress conditions. Grain yield is a complex trait that is governed by the coordinated action of several genetic and environmental factors. A number of genes and miRNAs have been identified to affect the grain productivity and yield. In this study, we identified the miRNAs that map to grain yield QTLs in rice. The expression variations of these miRNAs and their target transcript were studied across different tissues of three indica rice varieties with different grain morphology. The varieties used include the extra-long and slender grained Pusa Basmati 1 (PB1), medium grain sized IR64 and the short grained Pokkali (PK). The windows for miRNA target correlation were captured and their putative role in regulating rice grain yield is discussed.展开更多
The regulatory mechanisms of drought and salt-associated miRNAs have not been fully understood in Sorghum bicolor. In this study, we investigated the effect of salinity stress (200 and 300 mM NaCl) and drought stress ...The regulatory mechanisms of drought and salt-associated miRNAs have not been fully understood in Sorghum bicolor. In this study, we investigated the effect of salinity stress (200 and 300 mM NaCl) and drought stress at pre- and post-flowering stages on the expression pattern of small regulatory RNAs in six Sorghum genotypes using semi-quantitative reverse transcriptase PCR (RT-qPCR). The results indicated that both drought and salt stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought and salt stress in a different pattern among the six sorghum genotypes. miR156, miR167, miR168 and miR399 give different expressions levels compared to other studied miRNAs which may attribute to the adaption of sorghum to drought and salt stress and are good candidates for improving sorghum by transgenic technology.展开更多
文摘In the present study, we illustrate the strategy and protocol required to generate rice transgenics over-expressing the 21-nt form of Osa-miR820. The miR exists in two size variants of 21-nt and 24-nt so the natural precursor cannot be employed for the purpose of miR over-expression as the cellular machinery can process both size variants thereby masking the role of PTGS regulation. Hence, we adopted the artificial miR technology to specifically over-express the 21-nt species in the transgenics. During the course of experiments it was observed that the amiR constructs probably interfered with the regeneration of the transformed callus, necessitating protocol modifications. The results indicate the successful over-expression of the 21-nt miR species. These plants can serve as a useful source for the functional dissection of the role played by the 21-nt Osa-miR820 species. They will also be valuable in highlighting the importance for the existence of a dual mode of miR mediated target regulation.
文摘Plants face biotic and abiotic stresses during their entire life cycle, which leads to the loss in crop productivity. It has been shown that a relatively shorter exposure to heat stress, called priming, results in better adaptation of plants under subsequent stresses, which plants may face. While rice plants in nature often encounter high temperature stress conditions, the strategies to cope with those are poorly understood. We identified the involvement of microRNA pathways in the adaptation to heat stress (HS) at the physiological and molecular levels. It was observed that osa-miR169 levels are altered after HS and in response to light conditions. Its expression was also regulated by heat priming during anthesis and effectively responds to the successive exposure to high temperature stress during grain filling in rice. Osa-miR169 targets nuclear factor Y (NF-Y). We propose that osa-miR169: NF-Y regulatory module may be important for HS memory induced during high temperature priming and thus may serve to integrate stress responses with light regulated development. The future study in this direction will be useful to understand how plants acclimatize to the changing environment and thus help in generating stress tolerant crops.
文摘RNAi is an efficient surveillance machinery that plays a robust defensive role in shielding plant and animal hosts against viral infections. In counter-defense viruses encode suppressor proteins that have the ability to restrict the RNAi machinery to ensure successful systemic invasion. The B2 protein of insect Flock House Virus (FHV-B2) and AC2 protein of Mungbean Yellow Mosaic India Virus (MYMIV-AC2) are two well-characterized suppressors of RNAi, capable of reversing reporter gene silencing. In this study, we compared the strength of the two suppressors by assaying for the degree of RNAi reversion and the duration of sustaining the reversal in planta. The suppression activity was observed by assaying for GFP fluorescence at 3 dpi, 7 dpi and 14 dpi. The phenotypic observations were corroborated with small RNA Northern Blotting and semi-quantitative RT-PCR. The results indicate that suppressor strength of FHVB2 is comparable to MYMIV-AC2, although they are encoded by virus infecting host from two different eukaryotic kingdoms. This study will provide new insights to dissect the conservation in the RNAi pathways during the host-virus interactions.
文摘microRNAs (miRNAs) are key regulatory molecules that fine tune gene expression at the transcriptional and post-transcriptional levels through the RNA silencing pathways. They play an important role in regulating plant growth, development and response to a/biotic stress conditions. Grain yield is a complex trait that is governed by the coordinated action of several genetic and environmental factors. A number of genes and miRNAs have been identified to affect the grain productivity and yield. In this study, we identified the miRNAs that map to grain yield QTLs in rice. The expression variations of these miRNAs and their target transcript were studied across different tissues of three indica rice varieties with different grain morphology. The varieties used include the extra-long and slender grained Pusa Basmati 1 (PB1), medium grain sized IR64 and the short grained Pokkali (PK). The windows for miRNA target correlation were captured and their putative role in regulating rice grain yield is discussed.
文摘The regulatory mechanisms of drought and salt-associated miRNAs have not been fully understood in Sorghum bicolor. In this study, we investigated the effect of salinity stress (200 and 300 mM NaCl) and drought stress at pre- and post-flowering stages on the expression pattern of small regulatory RNAs in six Sorghum genotypes using semi-quantitative reverse transcriptase PCR (RT-qPCR). The results indicated that both drought and salt stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought and salt stress in a different pattern among the six sorghum genotypes. miR156, miR167, miR168 and miR399 give different expressions levels compared to other studied miRNAs which may attribute to the adaption of sorghum to drought and salt stress and are good candidates for improving sorghum by transgenic technology.
