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

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.