Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.

Rationale: Demyelination is a major component of white matter injury, characterized by oligodendrocyte (OL) death and myelin sheath loss, which result in memory loss and cognitive impairment in the context of ischemic stroke. Accumulating evidence has shown that OLs can be generated by the direct activation of defined transcription factors (TFs) in human induced pluripotent stem cells (hiPSCs); however, the rapid acquisition of single TF-induced OL progenitor cells (OPCs) as cell therapy for ischemic stroke remains to be thoroughly explored.

Summary: Bidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue‐specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue‐specific synthetic promoters. It is the first report on bidirectional green tissue‐specific promoters that could be efficiently applied in genetic engineering.

Brassica napus is a Cd hyperaccumulator, which is a serious threat to food and fodder safety. However, no related studies on developing Cd-safe B. napus have been reported yet. Here, we screened out a novel Cd uptake-related gene, AtCUP1, from the major facilitator superfamily in Arabidopsis thaliana. The mutation of AtCUP1 decreased Cd accumulation, both in roots and shoots of A. thaliana. Furthermore, the disruption of the AtCUP1 gene by the CRISPR/Cas9 system significantly reduced Cd accumulation in A. thaliana. Interestingly, the disruption of the BnCUP1 gene, an orthologous gene of AtCUP1, by the CRISPR/Cas9 system also diminished Cd accumulation in both roots and shoots of B. napus based on the hydroponics assay. Furthermore, for the field experiment, the Cd accumulations of BnCUP1-edited lines were reduced by 52% in roots and 77% in shoots compared to that of wild-type (WT) lines, and the biomass and yield of BnCUP1-edited lines increased by 42% and 47% of that of WT, respectively. Noteworthily, agronomic characteristics of B. napus were not apparently affected by BnCUP1-editing. Thus, BnCUP1-edited lines are excellent non-transgenic germplasm resources for reducing Cd accumulation without a distinct compromise in yield, which could be applied to agricultural production in Cd-contaminated soils.