Protocol for Artificial MicroRNA  Mediated Over-Expression of miR820  in Indica Rice

Sharma, Neha; Panchal, Sandeep; Sanan‐Mishra, Neeti

doi:10.4236/ajps.2015.612196

Cited by 11 publications

(6 citation statements)

References 41 publications

(43 reference statements)

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“…S5b-d), so that same amiRNA construct can also be used against other begomoviruses. The three designed amiRNAs (Table 1) were amplified by four sets of primers ( Supplementary Table S3) via overlapping PCR on the pNW55 vector backbone for obtaining pre-miRNA as given in Sharma et al 53 . The pre-amiRNA fragment was then cloned in pRT101, using XhoI and BamHI sites for directional cloning, under CaMV-35S promoter.…”

Section: Detection and Confirmation Of Rss Activitymentioning

confidence: 99%

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

Agarwal

Anand

et al. 2021

Sci Rep

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The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

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Section: Detection and Confirmation Of Rss Activitymentioning

confidence: 99%

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

Agarwal

Anand

et al. 2021

Sci Rep

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“…Another study revealed that the overexpression of the osa-miR7695 resulted in enhanced disease resistance to the fungal pathogen Magnaporthe oryzae , the cause of rice blast disease (Campo et al, 2013 ). Another recent study reported that Osa-miR820 plays a role in salt, high temperature, and drought stress responses (Sharma et al, 2015a ).…”

Section: Functional Roles Of Mirnasmentioning

confidence: 99%

Functional Roles of microRNAs in Agronomically Important Plants—Potential as Targets for Crop Improvement and Protection

et al. 2017

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MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression, mainly through cleavage and/or translation inhibition of the target mRNAs during or after transcription. miRNAs play important roles by regulating a multitude of biological processes in plants which include maintenance of genome integrity, development, metabolism, and adaptive responses toward environmental stresses. The increasing population of the world and their food demands requires focused efforts for the improvement of crop plants to ensure sustainable food production. Manipulation of mRNA transcript abundance via miRNA control provides a unique strategy for modulating differential plant gene expression and miRNAs are thus emerging as the next generation targets for genetic engineering for improvement of the agronomic properties of crops. However, a deeper understanding of its potential and the mechanisms involved will facilitate the design of suitable strategies to obtain the desirable traits with minimum trade-offs in the modified crops. In this regard, this review highlights the diverse roles of conserved and newly identified miRNAs in various food and industrial crops and recent advances made in the uses of miRNAs to improve plants of agronomically importance so as to significantly enhance crop yields and increase tolerance to various environmental stress agents of biotic-or abiotic origin.

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“…Therefore, artificial miR based strategy was adopted to specifically over-express the 21-nt length variants. The Osa-miR820 overexpressing transgenic lines (OX-820) were generated and confirmed, as described earlier [40].…”

Section: Resultsmentioning

confidence: 99%

“…The regulatory role of this miR on DRM2 transcripts was evident and the narrow windows of transcript regulation were captured. For gaining further insights into the biological significance of 21-nt species of Osa-miR820 in rice, artificial miR based over-expressing transgenic lines, OX-820 were generated [40]. In the present work, we report the phenotype and physiology of OX-820 under normal and saline conditions.…”

Section: Introductionmentioning

confidence: 99%

Osa-miR820 regulatory node primes rice plants to tolerate salt stress in an agronomically advantageous manner

Sharma

Kumar

Sanan-Mishra

2021

Preprint

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Plant microRNAs (miRs) play an important role in regulating gene expression under normal and stressful environments. Here we report the functional implications on the role of Osa-miR820, which can be grouped as a young, rice-specific miR. It is a member of the class II transposon-derived small RNA family and is processed as 21-nt and 24-nt length variants, respectively. Size of the miR820 family varies from 1 to 16 across the Oryza AA genomes. The 21–nt Osa-miR820 negatively regulates a de novo methylase, OsDRM2 (domains rearranged methyl transferase) that prevents methylation of the CACTA transposon loci in the rice genome. In an earlier report we have detailed the expression profiles of Osa-miR820 and its target in abiotic stress responses using rice varieties exhibiting varying response to salt stress. In this study, artificial miR based approach was employed to specifically overexpress 21-nt Osa-miR820 in rice plants (OX-820). These plants exhibited enhanced vigour, ~25-30% increase in the number of spikelets per panicle and increased grain filling, under normal and salt stress conditions. The OX-820 lines showed a better water use efficiency and higher proline accumulation under salt stress. These plants can serve as a useful source for dissecting the molecular machinery governed by Osa-miR820:DRM2 node to prime tolerance to salt stress in an agronomically advantageous manner.

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Protocol for Artificial MicroRNA Mediated Over-Expression of miR820 in Indica Rice

Cited by 11 publications

References 41 publications

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

Functional Roles of microRNAs in Agronomically Important Plants—Potential as Targets for Crop Improvement and Protection

Osa-miR820 regulatory node primes rice plants to tolerate salt stress in an agronomically advantageous manner

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