Towards genetic modification of the lignin biosynthetic pathway in interior spruce (Picea glauca x engelmanni complex).

摘要

Although the lignin biosynthetic pathway has been altered successfully in angiosperm species via genetic engineering approach, this has not yet been achieved in gymnosperm species. Therefore, the goal of my thesis research was to apply the transgenic approach to economically important interior spruce (Picea glauca × engelmanni complex).; In the first half of my thesis, the poplar PAL2-GUS fusion gene was introduced into hybrid poplar (Populus tremula × P. alba) and interior spruce in order to evaluate the potential use of this promoter for directing xylem-specific gene expression. In transgenic poplar, the poplar PAL2 promoter directed the expression of the GUS gene in the tissues associated with synthesis of phenolic compounds (epidermal/subepidennal cells) and in the tissues associated with lignin synthesis (xylem and phloem cells). In contrast, in transgenic spruce, the activity of the poplar PAL2 promoter was detected only in the tissues associated with lignin and suberin synthesis. The differences in the activity of the poplar PAL2 promoter between the two hosts suggest that the gene regulation system that leads to the synthesis of essential structural components such as lignin and suberin is more likely to be conserved than that leading to the synthesis of specialized phenolic compounds.; The second half of this thesis investigated a contribution of coniferin β-glucosidase (CG) in lignin synthesis in spruce. CG is believed to be involved in the last steps of the lignin biosynthetic pathway by releasing the lignin precursor, coniferyl alcohol, from its glucosides form, coniferin, before polymerization in the cell wall. An antisense construct against the CG gene was prepared using the lodgepole pine CG cDNA sequence and was introduced into interior spruce. Among 45 antisense lines examined, no transgenic lines contained reduced levels of endogenous CG mRNA levels. The failure of the antisense CG gene to cause inhibitory effects on the endogenous CG expression levels in transgenic spruce could be attributed to low expression of the antisense gene, or to insufficient sequence homology between the antisense and target CG sequences. To my knowledge, this is the first study to employ an antisense approach in a gymnosperm species.