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Research Article Open access CC BY 4.0

Construction of an Agrobacterium Mediated RNAI Genetic Transformation Vector Targeting the Replicase Gene of Indian cassava mosaic virus and Evaluation of Their Transformation Ability in Cassava Immature Leaf Lobes

T. Anuradha, P. Prabha

Biotechnology Journal International · pp. 17–24 · Published 25 Mar 2021

10.9734/bji/2021/v25i230135

Abstract

Cassava mosaic virus is one of the major problems affecting cassava industry in India. Currently there are no effective strategy to completely protect cassava from cassava mosaic viruses. In order to attain cassava mosaic virus resistance RNAi vectors targeting the replicase gene of Indian cassava mosaic virus is constructed in this study. Their efficiency to transform cassava immature leaf lobes were also studied here. Replicase gene of Indian cassava mosaic virus in Tamilnadu are cloned and sequenced. Conserved domains are identified and sub cloned to CSIRO RNAi vector system and transformation studies are done in immature cassava leaves. Two different RNAi vectors were constructed, utilizing a conserved 440bp of 5’ end of ICMV Rep (AC1) gene which also corresponds to a part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of ICMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL. After cloning into pHANNIBAL, the cloned RNAi gene cassettes of ICMV is released and cloned into the binary vector, pART27, which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as a selection agent in cassava genetic transformation, RNAi–Rep gene cassettes of ICMV were cloned into pCAMBIA1305.2. These constructs were named pICR1 and pICR2 respectively. The Genetic transformation studies in cassava leaves done using pICR2 vector could generate PCR positive plants. An agrobacterium mediated replicase RNAi vector is developed and that can be transformed into cassava immature leaf lobes. Their efficiency to silence the Indian cassava mosaic virus should be studied further.

RNAi constructs Indian cassava mosaic virus replicase binary vector genetic transformation.

References (17)

  1. 1 Construct design for efficient, effective and high‐throughput gene silencing in plants [DOI]
  2. 2 Geminiviruses: Models for Plant DNA Replication, Transcription, and Cell Cycle Regulation [DOI]
  3. 3 Short Interfering RNA Accumulation Correlates with Host Recovery in DNA Virus-Infected Hosts, and Gene Silencing Targets Specific Viral Sequences [DOI]
  4. 4 Effect of Temperature on Geminivirus-Induced RNA Silencing in Plants [DOI]
  5. 5 Development and application of transgenic technologies in cassava [DOI]
  6. 6 Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava [DOI]
  7. 7 Characterisation of Sri Lankan Cassava Mosaic Virus and Indian Cassava Mosaic Virus: Evidence for Acquisition of a DNA B Component by a Monopartite Begomovirus [DOI]
  8. 8 RNAi targeting of DNA virus in plants [DOI]
  9. 9 Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication [DOI]
  10. 10 Infectivity of the cloned geminivirus genome requires sequences from both DNAs [DOI]
  11. 11 The Role of AV2 (“Precoat”) and Coat Protein in Viral Replication and Movement in Tomato Leaf Curl Geminivirus [DOI]
  12. 12 Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes [DOI]
  13. 13 Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs [DOI]
  14. 14 Production of transgenic cassava (Manihot esculenta Crantz) plants by particle bombardment using luciferase activity as selection marker [DOI]
  15. 15 Transactivation of Geminivirus AR1 and BR1 Gene Expression by the Viral AL2 Gene Product Occurs at the Level of Transcription [DOI]
  16. 16 Short Interfering RNA Accumulation Correlates with Host Recovery in DNA Virus-Infected Hosts, and Gene Silencing Targets Specific Viral Sequences [DOI]
  17. 17 Food and Agriculture Organization of the United Nations [DOI]

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