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Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

ABSTRACT: We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlets were identified. Real-time quantitative PCR (RT-qPCR) revealed that the copy number of cry1Ac gene in the transgenic lines varied from 1 to 148. ELISA analysis showed that Cry1Ac protein levels in 7 transgenic lines ranged from 0.85 ?g/FWg to 70.92 ?g/FWg in leaves and 0.04 ?g/FWg to 7.22 ?g/FWg in stems, and negatively correlated to the rate of insect damage that ranged from 36.67% to 13.33%, respectively. Agronomic traits of six transgenic sugarcane lines with medium copy numbers were similar to the non-transgenic parental line. However, phenotype was poor in lines with high or low copy numbers. Compared to the non-transgenic control plants, all transgenic lines with medium copy numbers had relatively equal or lower sucrose yield and significantly improved sugarcane borer resistance, which lowered susceptibility to damage by insects. This suggests that the transgenic sugarcane lines harboring medium copy numbers of the cry1Ac gene may have significantly higher resistance to sugarcane borer but the sugarcane yield in these lines is similar to the non-transgenic control thus making them superior to the control lines.

SUBMITTER: Gao S

PROVIDER: S-EPMC4836700 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

Gao Shiwu S Yang Yingying Y Wang Chunfeng C Guo Jinlong J Zhou Dinggang D Wu Qibin Q Su Yachun Y Xu Liping L Que Youxiong Y

PloS one 20160419 4

We developed sugarcane plants with improved resistance to the sugarcane borer, Diatraea saccharalis (F). An expression vector pGcry1Ac0229, harboring the cry1Ac gene and the selectable marker gene, bar, was constructed. This construct was introduced into the sugarcane cultivar FN15 by particle bombardment. Transformed plantlets were identified after selection with Phosphinothricin (PPT) and Basta. Plantlets were then screened by PCR based on the presence of cry1Ac and 14 cry1Ac positive plantlet ...[more]

PMID: 27093437

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Project description:BackgroundGenetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood.ResultsTo investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes.ConclusionsThis study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Project description:The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Dataset Information

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

Publications

Transgenic Sugarcane with a cry1Ac Gene Exhibited Better Phenotypic Traits and Enhanced Resistance against Sugarcane Borer.

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