The application of the ‘Gene-deletor’ technology in banana

Banana (Musa spp.) is an important tropical crop. Banana industry is under biotic and abiotic stresses such as Fusarium wilt, typhoon, cold stress. Genetic engineering offers a powerful strategy to create germplasm of banana with enhanced resistance. The safety of genetically modified organisms has become a bottleneck restricting the popularization and application of genetically modified technology. In this study, a candidate promoter, LEAFY (LFY) for expression and flower initiation in Arabidopsis, was cloned and constructed to ‘Gene-deletor’ vector. Histochemical β-glucuronidase (GUS) staining results showed that the ‘Gene-deletor’ vector driven by LFY promoter could lead to 88.5% excision efficiency from Arabidopsis seeds based on more than 200 T3 progeny examined per event. GUS staining was found to be partially negative in transgenic bananas, however, polymerase chain reaction could still detect the presence of large fragments of the vector. These results suggest that although LFY promoter could not completely drive the ‘Gene-deletor’ vector to achieve the effect of complete elimination of exogenous gene in bananas, its efficiency of eliminating exogenous gene laid a theoretical foundation for cloning banana fruit-specific promoters, that is, ‘non-transgenic’ GM bananas.

DNA methylation subpatterns at distinct regulatory regions in human early embryos

DNA methylation has been investigated for many years, but recent technologies have allowed for single-cell- and single-base-resolution DNA methylation datasets and more accurate assessment of DNA methylation dynamics at the key genomic regions that regulate gene expression in human early embryonic development. In this study, the region from upstream 20 kb to downstream 20 kb of RefSeq gene was selected and divided into 12 distinct regions (up20, up10, up5, up2, 5'UTR, exon, intron, 3'UTR, down2, down5, down10 and down20). The candidate promoter region (TSS ± 2 kb) was further divided into 20 consecutive subregions, which were termed ‘bins’. The DNA methylation dynamics of these regions were systematically analysed along with their effects on gene expression in human early embryos. The dynamic DNA methylation subpatterns at the distinct genomic regions with a focus on promoter regions were mapped. For the 12 distinct genomic regions, up2 and 5'UTR had the lowest DNA methylation levels, and their methylation dynamics were different with other regions. The region 3'UTR had the highest DNA methylation levels, and the correlation analysis with gene expression proved that it was a feature of transcribed genes. For the 20 bins in promoter region, the CpG densities showed a normal distribution pattern, and the trend of the methylated CpG counts was inverse with the DNA methylation levels, especially for the bin 1 (downstream 200 bp of the TSS). Through the correlation analysis between DNA methylation and gene expression, the current study finally revealed that the region bin −4 to 6 (800 bp upstream to 1200 bp downstream of the TSS) was the best candidate for the promoter region in human early embryos, and bin 1 was the putative key regulator of gene activity. This study provided a global and high-resolution view of DNA methylation subpatterns at the distinct genomic regions in human early embryos.

Cloning and characterization of a cold inducible Pal promoter from Fagopyrum tataricum

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in biosynthesis pathway of flavonoids and plays an important role in plant stress resistance. In this study, the 5’ flanking region of phenylalanine ammonia-lyase gene was isolated from Fagopyrum tataricum by thermal asymmetric interlaced PCR method, named PFtPal (GenBank: KF463139). To investigate the functional properties of PFtPal, we constructed a series of plant expression vectors that contained different promoter fragments resulting from nest deletions and had successfully transformed them into tobacco leaves by Agrobacterium tumefaciens. Histochemical assay of GUS suggested that PFtPal could drive GUS gene expression in leaves and roots, while GUS activity was not detected in the stem. In addition, the region of −274 bp to −1 bp was enough to drive normal expression of GUS gene. Low temperature treatment of transgenic tobacco plants demonstrated that PFtPal conferred cold-induced expression. Taken together, our study will help to better understand the Pal promoter, and provides a candidate promoter for molecular breeding in Fagopyrum plants.