Development and Evaluation of the Cotton Leaf Curl Kokhran Virus-Burewala Bidirectional Promoter for Enhanced Cry1Ac Endotoxin Expression in Bt Transgenic Cotton

Abstract

Fluctuation in Cry1Ac endotoxin levels expressed in transgenic Bacillus thuringiensis (Bt) cotton (Gossypium hirsutum L.) can result in a variation in efficacy throughout the growing season. Here, a green tissue-specific strong promoter of the cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) C1 gene is reported that can direct consistently high levels of Cry1Ac endotoxin expression in transformed cotton plants. The objective of this study was to investigate the capacities of the CLCuKoV-BuC1 promoter to drive transcription of Cry1Ac and stably express endotoxin in mature leaves and bolls of transgenic cotton plants, compared to the traditional CaMV35S promoter. The Cry1Ac gene expression cassettes were constructed under the control of a bidirectional promoter and transformed into cotton ‘MNK-786′. The expression of Cry1Ac constructs was evaluated in transient and stable expression systems using Nicotiana tabacum ‘Rustica’ and cotton plants, respectively. Accumulation of the Cry1Ac expressed in two resultant transgenic cotton plants harboring the constructs driven by the CLCuKoV-BuC1 and CaMV35S promoter, respectively, was analyzed using a commercially available enzyme-linked immunosorbent assay. In leaves and bolls of two cotton plants shown to express CLCuKoV-BuC1-Cry1Ac (CLCuV-Ac), the Cry1Ac protein accumulated at 400 and 300 ng g−1 per fresh tissue weight, respectively, whereas no toxin was detectable in the roots. In contrast, CaMV35S-Cry1Ac transgenic cotton plants accumulated three times less Cry1Ac protein than those transformed with CLCuV-Ac. Results indicate that the greatest amount of Cry1Ac endotoxin accumulated in transgenic cotton when expression was driven by the CLCuKoV-BuC1 compared to the CaMV35S promoter. Thus, the CLCuKoV-BuC1 promoter offered more robust transgene expression in cotton plants than the traditional CaMV35S promoter. The newly validated CLCuV-Ac promoter of begomoviral origin offers an exciting alternative as a robust promoter for genetic engineering of cotton and other plants.

In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava

“…The DNA-A genome of begomoviruses possesses a LIR that functions as a bidirectional promoter, essential for replication and expression of the viral genes, AC1 (Rep) and AV1 (CP) [ 67 , 68 ]. The miRanda algorithm predicted hybridization of mes-miR393 (a, b, c, and d) from the common locus 130 in the LIR of the ICMV-Ker genome ( Figure 2 A and Table 2 ).…”

The regulatory landscape of genetically engineered crops in Pakistan

“…Pakistan has not introduced any legislation for the commercial production of any genome‐edited crop out of the countries in this group. In Pakistan, many GM crops are produced as R&D in research institutes, only cotton is marketed commercially, and is used by the farmers (Ashraf, Shahid, Rao, Brown, & Husnain, 2022; Shad et al, 2022). The Pakistan Biosafety Rules 2005 by the Pakistan Environmental Protection Act (EPA), the Intellectual Property Organization of Pakistan Act of 2012, the Seed Amendment Act of 2015, and the Plant Breeders' Rights Act (PBR) of 2016 are the backbone of Pakistan's legal framework for agricultural biotechnology.…”

“…farmers(Ashraf, Shahid, Rao, Brown, & Husnain, 2022;Shad et al, 2022). The Pakistan Biosafety Rules 2005 by the Pakistan Environmental Protection Act (EPA), the Intellectual Property Organization of Pakistan Act of 2012, the Seed Amendment Act of 2015, and the Plant Breeders' Rights Act (PBR) of 2016 are the backbone of Pakistan's legal framework for agricultural biotechnology.…”

Genome-Wide Identification of Cotton MicroRNAs Predicted for Targeting Cotton Leaf Curl Kokhran Virus-Lucknow

“…Large intergenic region (LIR) drives the transcriptional regulation of the V1 and C1 ORFs of CLCuKoV [20,21,84]. Four distinct prediction algorithms (miRanda, RNA22, psRNATarget and RNAhybrid) predicted hybridization binding site of ghr-miR2950 at consensus genomic locus 82 of CLCuKoV targeting LIR (Figure 3 A-D).…”

“…The monopartite genome of a CLCuKoV encodes six proteins. The transcription and regulation of these proteins are governed from a large intergenic region (LIR) using bidirectional mode of transcription [12,20,21]. The plus (+) virion-sense (VS) and negative (-) complementary-sense (CS) strands encode ORFs V1 and V2, and ORFs C1, C2, C3 and C4, respectively [15,18,22].…”