ABSTRACT
MAIN CONCLUSION: The sorghum soluble acid invertase gene SbSAI-2 was cloned and the function verified in Pichia pastoris and rice, showing the SbSAI-2 affects composition and content of sugar in stem juice. Sugar metabolism is one of the most important metabolic processes in plants, in which soluble acid invertase plays a key role. However, the structure and function of the soluble acid transferase gene in sorghum are still fully unclear. In this study, SbSAI-2 was cloned from the sorghum variety BTx623, and two transcripts were found through sequence analysis, with only one transcript translated into an active protein. There is 72% homology between SbSAI-2 and OsVIN2. The construction of Osvin2 mutant lines and SbSAI-2-1 overexpression lines in Oryza sativa L. japonica. cv. Nipponbare were produced to clarify the invertase functionality. While the invertase activity in the stem of the Osvin2 mutant line was reduced, with no significant difference (P > 0.05), and the contents of fructose and glucose in stem tissue did not change significantly (P > 0.05), and the content of sucrose increased by 38.89% (P < 0.01). In SbSAI-2-1 overexpression lines, the invertase activity in stem was increased by more than 20 times (P < 0.01). The contents of glucose and fructose in stem tissues were increased by two and three times, respectively (P < 0.01), while the content of sucrose was significantly decreased, which was below the detection limit (P < 0.01). This study indicated that SbSAI-2 is a key enzyme related to sucrose metabolism and affects the composition and content of sugar in stems. The result provided further the gene function verification and laid a foundation for the development of molecular markers.
Subject(s)
Oryza , Sorghum , Cloning, Molecular , Edible Grain , Oryza/genetics , Sorghum/genetics , Sucrose , beta-Fructofuranosidase/geneticsABSTRACT
The potential impact of transgene escape on the environment and food safety is a major concern to the scientists and public. This work aimed to assess the effect of intein-mediated gene splitting on containment of transgene flow. Two fusion genes, EPSPSn-In and Ic-EPSPSc, were constructed and integrated into N. tabacum, using Agrobacterium tumefaciens-mediated transformation. EPSPSn-In encodes the first 295 aa of the herbicide resistance gene 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) fused with the first 123 aa of the Ssp DnaE intein (In), whereas Ic-EPSPSc encodes the 36 C-terminal aa of the Ssp DnaE intein (Ic) fused to the rest of EPSPS C terminus peptide sequences. Both EPSPSn-In and Ic-EPSPSc constructs were introduced into the same N. tabacum genome by genetic crossing. Hybrids displayed resistance to the herbicide N-(phosphonomethyl)-glycine (glyphosate). Western blot analysis of protein extracts from hybrid plants identified full-length EPSPS. Furthermore, all hybrid seeds germinated and grew normally on glyphosate selective medium. The 6-8 leaf hybrid plants showed tolerance of 2000 ppm glyphosate in field spraying. These results indicated that functional EPSPS protein was reassembled in vivo by intein-mediated trans-splicing in 100% of plants. In order to evaluate the effect of the gene splitting technique for containment of transgene flow, backcrossing experiments were carried out between hybrids, in which the foreign genes EPSPSn-In and Ic-EPSPSc were inserted into different chromosomes, and non-transgenic plants NC89. Among the 2812 backcrossing progeny, about 25% (664 plantlets) displayed glyphosate resistance. These data indicated that transgene flow could be reduced by 75%. Overall, our findings provide a new and highly effective approach for biological containment of transgene flow.
Subject(s)
Genetic Techniques , Nicotiana/genetics , Transgenes/genetics , 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics , Blotting, Western , Gene Flow , Glycine/analogs & derivatives , Glycine/toxicity , Herbicide Resistance/genetics , Homozygote , Inteins/genetics , Peptides/metabolism , Plants, Genetically Modified , Recombinant Fusion Proteins/metabolism , Nicotiana/growth & development , Trans-Splicing , Transformation, Genetic , GlyphosateABSTRACT
A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.
