Ubiquitin promoter�Cterminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.     

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants

Key Message

Differential expression of mi-RNAs targeting developmental processes and progressive downregulation of repeat-associated siRNAs following genome merger and genome duplication in the context of allopolyploid speciation in Spartina.

Abstract

The role of small RNAs on gene expression regulation and genome stability is arousing increased interest and is being explored in various plant systems. In spite of prominence of reticulate evolution and polyploidy that affects the evolutionary history of all plant lineages, very few studies analysed RNAi mechanisms with this respect. Here, we explored small RNAs diversity and expression in the context of recent allopolyploid speciation, using the Spartina system, which offers a unique opportunity to explore the immediate changes following hybridization and genome duplication. Small RNA-Seq analyses were conducted on hexaploid parental species (S. alterniflora and S. maritima), their F1 hybrid S. x townsendii, and the neoallododecaploid S. anglica. We identified 594 miRNAs, 2197 miRNA-target genes, and 3730 repeat-associated siRNAs (mostly targeting Class I/Copia-Ivana- Copia-SIRE and LINEs elements). For both mi- and ra-siRNAs, we detected differential expression patterns following genome merger and genome duplication. These misregulations include non-additive expression of miRNAs in the F1 hybrid and additional changes in the allopolyploid targeting developmental processes. Expression of repeat-associated siRNAs indicates a strengthen of transposable element repression during the allopolyploidization process. Altogether, these results confirm the central role small RNAs play in shaping regulatory changes in naturally formed recent allopolyploids.

     

Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter

The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.     

Colorectal Cancer Genetic Heterogeneity Delineated by Multi-Region Sequencing

Intratumor heterogeneity (ITH) leads to an underestimation of the mutational landscape portrayed by a single needle biopsy and consequently affects treatment precision. The extent of colorectal cancer (CRC) genetic ITH is not well understood in Chinese patients. Thus, we conducted deep sequencing by using the OncoGxOne^™ Plus panel, targeting 333 cancer-specific genes in multi-region biopsies of primary and liver metastatic tumors from three Chinese CRC patients. We determined that the extent of ITH varied among the three cases. On average, 65% of all the mutations detected were common within individual tumors. KMT2C aberrations and the NCOR1 mutation were the only ubiquitous events. Subsequent phylogenetic analysis showed that the tumors evolved in a branched manner. Comparison of the primary and metastatic tumors revealed that PPP2R1A (E370X), SETD2 (I1608V), SMAD4 (G382T), and AR splicing site mutations may be specific to liver metastatic cancer. These mutations might contribute to the initiation and progression of distant metastasis. Collectively, our analysis identified a substantial level of genetic ITH in CRC, which should be considered for personalized therapeutic strategies.