Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane

Abstract

Sugarcane mosaic virus (SCMV) is a plant pathogenic virus of the family Potyviridae that causes chlorosis, stunting and significantly reduced sugar productivity in sugarcane. Pathogen-derived resistance is a method used to develop SCMV-resistant sugarcane by overexpression of viral DNA. In this study, the gene encoding the coat protein (CP) of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. Nucleotide sequence analysis of amplified cDNA indicated that the 998-bp-long cDNA, termed ScMVCp cDNA, codes for the CP of SCMV from the PS881 isolate. The ScMVCp cDNA was inserted into the binary vector pRI101-ON with two constructs, a full nucleotide sequence (p927) and a sequence coding for N-terminally truncated protein (p702). The constructs were then introduced into sugarcane using Agrobacterium-mediated transformation. Southern blot analysis showed a single hybridized DNA copy inserted into the genome of transgenic sugarcane lines. The inserted genes were expressed at both the RNA transcript and protein levels in the transgenic sugarcane. The highest expression was found in transgenic lines 10, 11 and 13 from the p927 construct. Artificial infection by the virus showed that p927 generated a higher resistance to virus compared with p702. This resistance was passed on to the second generation of transgenic sugarcane with 100 and 20-40% levels of resistance in the p927 and p702 transgenic lines, respectively. This report shows that the full sequence of the CP gene is required to disrupt viral assembly and packaging, thereby generating resistance to SCMV infection.

Overexpression of Sucrose Phosphate Synthase Enhanced Sucrose Content and Biomass Production in Transgenic Sugarcane

“…The amplified cDNA was digested with the SpeI (XbaI compatible) and inserted into the XbaI site of GUS-removed pBI121 plasmid. Sugarcane in vitro shoots were used as explant for Agrobacterium transformation according to the method previously described [43]. The sugarcane shoot was prepared by micropropagation of meristematic apical tissue isolated from 4 to 5 months of sugarcane growth in the field of Bululawang (BL) cultivars.…”

“…Genomic DNA was isolated from 3 g sugarcane leaves, as previously described [43], and stored at −20 • C until analysis. The presence of the inserted gene of interest was analyzed by PCR using the genomic DNA and a pair of primers for the detection of nptII gene ( Table 3).…”

Development of Allergenicity and Toxicity Assessment Methods for Evaluating Transgenic Sugarcane Overexpressing Sucrose–Phosphate Synthase

“…Sugarcane is a major crop that produces sugar in tropical regions, and transgenic sugarcane has been recently developed for drought stress tolerance [9,10], pest resistance [11,12], virus resistant [13,14], and increased crop yield by introducing a gene for sucrose-phosphate synthase (SPS; EC 2.4.1.14) (under review). Several tests for allergenicity and toxicity have to be evaluated in transgenic sugarcane overexpressing gene SPS, since there is no report for the evaluation in transgenic sugarcane.…”

Cloning, transformation and expression of cell cycle-associated protein kinase OsWee1 in indica rice (Oryza sativa L.)

“…Southern blot analysis was performed by method previously described [19]. Twenty μg of the genomic DNA was digested with the restriction enzymes BamHI (Promega) at 37 °C overnight.…”