Vectors for RNAi Technology in Poplar

Abstract

The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar. A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the beta-glucuronidase (GUS) reporter gene system. These gene cassettes are driven by the CaMV-35S promoter. To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants. Agrobacterium-mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene. From these results we conclude that RNAi is also functional in poplar.

The Current Status and Development of Insect-Resistant Genetically Engineered Poplar in China

“…RNAi technology had been successfully applied in the study of various types of insects in recent years and many suitable target genes have emerged, such as chitin synthesis-related enzymes (chitin synthase and chitinase gene) (Arakane et al, 2005; Chen et al, 2008), hormones and receptors related to growth and development (epidermal hormones receptors EcR and juvenile hormones) (Huang et al, 2013), enzymes that modulate energy metabolism (proton metabolized V-ATPase and cytochrome P450 enzymes that metabolize toxic substances) (Tang et al, 2012; Kotwica-Rolinska et al, 2013), as well as neuromodulation pathways receptor and related enzymes (Agrawal et al, 2013). RNAi-mediated down-regulation of poplar had been studied and proved that RNAi is also functional in poplar (Mryer et al, 2004; Bi et al, 2015). …”

Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees

“…Generation of constructs for PtAO2 and PtABA3 knock down transformants was accomplished according to [36]. For the ABA3-RNAi constructs two exons of ptaba3 (LG_VII 7136650-7136823 (174 bp) and LG_VII 7137117-7137323 (207 bp)) were combined in a fusion PCR using P. trichocarpa DNA as template.…”

“…For AO-RNAi constructs a 410 bp fragment of the ptao2 sequence was amplified from P. trichocarpa genomic DNA (Primer : Table S1). In each case the resulting fragments were used as sense and antisense fragments and exchanged the gusA fragments in the existing RNAi construct from [36] for creating a ptaba3 or ptao2 RNAi cassette, respectively. After generation of the chimeric gene cassettes cloning into the binary vector pBIN19 was achieved via HindIII restriction sites [38].…”

“…Gene constructs for promoter analyses contained the putative promoter sequences of ptaba3 or ptao2 which were fused to β-glucuronidase (gusA). RNAi constructs were generated using the RNAi-cassette obtained from Meyer et al (2004). ABA3 over-expressors were constructed using a combination of the cauliflower 35S-promoter, the enhanced green fluorescent protein (gusA) and the Arabidopsisaba3 sequence.…”

“…Because poplar gene knockouts are difficult to generate due to long generation times we used RNAi-constructs [36] for decreasing AO enzyme activity and consequently, decreasing endogenous ABA levels. For aba3 and ao promoter analyses, β-glucuronidase (GUS) fusion approaches were used.…”