Gene silencing studies in the gymnosperm species Pinus radiata

Abstract

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.

Suppression of 4-Coumarate-CoA Ligase in the Coniferous GymnospermPinus radiata

“…RNAi in plants is based on both cell-autonomous and non-cell-autonomous processes (Shimamura et al, 2007), which might allow for differential responses to occur at the cellular level. RNAi can also be affected by developmental processes in pine (Wagner et al, 2005(Wagner et al, , 2007. These and other factors, therefore, might affect the phenotype at the cellular level in wood.…”

Gene silencing: concepts, applications, and perspectives in woody plants

“…In TGS, silenced transgenes coding regions and promoters are densely methylated (Kooter et al, 1999). It is also proposed that the increase in DNA methylation possibly induces formation of heterochromatin, which is associated to TGS (Ye et al, 1996;Wassenegger & Pélissier, 1998;Wassenegger, 2000). DNA methylation promotes protein binding that recognizes methylated cytosine, leading to chromatin remodeling (Alberts et al, 2002), thus avoiding the binding of transcription factors (Kooter et al, 1999).…”

“…(Ko et al, 1998;Dandekar et al, 2004;Viss et al, 2003;Broothaerts et al, 2004;Gilissen et al, 2005;Teo et al, 2006), Pinus radiata D. Don. (Moller et al, 2005;Tang et al, 2005a;2005b;Wagner et al, 2005;Tang et al, 2006), Juglans regia L. (Escobar et al, 2002), Coffea canephora (Ogita et al, 2003), Eucalyptus sp. (Valério et al, 2003;Sassaki et al, 2005), Caryca papaya (Tennant et al, 2001), and Actinidia deliciosa (A.Chev.)…”

“…Gene silencing (GS) is defined as a molecular process involved in the down regulation of specific genes, and probably evolved as a genetic defense system against viruses and invading nucleic acids (Brigneti et al, 1998;Voinnet et al, 2000;Waterhouse et al, 2001;Wassenegger, 2002). Currently, there are several routes of GS identified in plants, such as: posttranscriptional gene silencing or RNA interference (PTGS or RNAi) , transcriptional gene silencing , microRNA silencing (Bartel, 2004), and virus induced gene silencing (Burch-Smith et al, 2004).…”

Genetic Engineering in Conifer Plantation Forestry

“…The more recent application of commercially important genes, such as insect and herbicide resistance and lignin biosynthesis genes, demonstrate that conifer biotechnology is now entering a phase of potential commercial deployment in plantation forestry (BISHOP HURLEY et al, 2001;CHARITY et al, 2005;GRACE et al, 2005;TANG et al, 2003;WAGNER et al, 2005).…”