Isolation and characterization of an RNA polymerase III encoded gene of Pinus radiata and its use in pine transformation

Abstract

Several promoters such as the cauliflower mosaic virus 35S promoter (CaMV 35S) and its enhanced version, pEMU, the maize ubiquitin promoter, the alcohol dehydrogenase promoter (Adh) and rice actin promoter (Act1) are currently used in Pinus radiata (pine) transformation. These heterologous promoters were adopted for pine transformation for want of an endogenous promoter tailored specifically for the needs of pine. These promoters may not perform to the same extent in pine as in their homologous systems due to differences in quality and/or quantity of regulatory factors. Secondly, because of their heterologous origin, these promoters are open to silencing mechanisms that operate in plants against invasive DNA [Matzke & Birchler, 2005]. This could result in inactivation of these promoters at any time during the 30-year growth period of transformed pine which this poses a real threat to a forestry industry based on transgenic pine. A pine promoter on the other hand, being endogenous, is less prone to silencing. In addition, confidence in its longevity (continued expression) can be easily established even before using it in transformation. The aim of this study was to isolate and validate pine promoters that can be used in pine transformation. As only a few pine sequences were available in the public domain for gene discovery in pine (at the beginning of this study), heterologous sequence information was used to screen the pine genome or its transcriptome for orthologs with desirable expression features. The investigation proceeded along two lines. In the first approach, a putatively desirable gene was isolated and the expression profile of its promoter was then validated. This led to the characterization of 5Spr20, a pine SS rDNA paralog. 5Spr20 differs from all published SS rDNA sequence& dnd is therefore a novel pine gene. Analyses of its sequence using bioinformatics revealed that it is capable of initiating biologically active transcripts and 5Spr20 is therefore a functional gene. A recombinant 5Spr20 promoter consisting of the coding region and the immediately upstream region downregulated gus reporter activity by 90% by antisense activity in transient expression studies in pine embryogenic cells. In stable expression studies, a 5Spr20 promoter-driven shDNA construct targeting gus completely silenced reporter activity in the model plant Nicotiana benthamiana. The 5Spr20 promoter appears to hold great promise for use in pine functional genomics and in gene downregulation applications. In the second line of investigation, the expression profiles of pine orthologs of known heterologous genes were validated prior to gene isolation. Two pine genes that were identified as promising candidates are pine tDNAMet-l and an actin paralog pine, ActX. Both genes were strongly expressed in all vegetative tissues of pine. Several PCR-based methods were used to clone the upstream regions (containing putative promoter elements) but all attempts ended in failure, which is attributed to the presence of pseudogenes and regions homologous to walking/sequencing primers among paralogs. The pine transcriptome was also screened unsuccessfully for ortholgs of desirable heterologous candidate genes like the ribosomal protein genes MsRL5 of Medicago sativa and AtL 18 of Arabidopsis thaliana and genes for the second largest subunit of RNA polymerase II, gene T13794 and actin-2 of A. thaliana. Sequence heterogeneity, cell-specific expression and low transcript abundance are possible reasons for not being able to detect pine orthologs of these candidate genes in expression screens.