Establishment of Pale Yellow 'Golden Pothos' Plant-based Transgenic System for Studying Nuclear Genes Involved in Chloroplast Biogenesis.

摘要

Chloroplast has many functions and is a critical organelle for plants and green algae. Chloroplasts are responsible for photosynthesis and the synthesis of amino acids, lipids, plant hormones and many other metabolites. However, the number of genes required for chloroplast biogenesis and function remains unclear. Chloroplasts develop from proplastids by a series of steps, which require genes from both the nuclear and chloroplast genomes. It is estimated that chloroplast biogenesis and function require 2,500 to 3,500 proteins. Although chloroplast requires so many proteins, its genome only has capacity to encode less than 100 proteins, while over 95% of proteins are encoded by nuclear genes, which are synthesized in the cytosol and then imported into proplastids for chloroplast biogenesis. Given that chloroplast proteins are encoded by two separate genomes, understanding the number of genes and their coordinated expression during chloroplast biogenesis is both of great fundamental and practical importance. To study these nuclear genes coding for chloroplast proteins, a novel pale yellow plant based alcA inducible system has been proposed. In this system, regenerated pale yellow plants from the naturally variegated 'Golden Pothos' were used whose color defection had been confirmed resulting from low expression of nuclear gene, EaZIP. This gene encodes for Mg-protoporphyrin IX monomethyl ester cyclase, which is an important enzyme in the chlorophyll biosynthesis pathway. To confirm functionality of AlcA system, EaZIP driven by an inducible promoter was expressed in tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. Induction of the tobacco explants proved that the alcA system is not only functional, but highly sensitive to ethanol induction. Given that the genetic cassette functions in tobacco, the genetic cassette was transformed into pale yellow 'Golden Pothos'. The transgene in pale yellow 'Pothos' has yet to be confirmed in transgenic pale yellow Pothos, due to slow growth and oxidation. Once slow growth and the oxidation issues are resolved the novel inducible assay system can possibly be used to study those nuclear encoding chloroplast proteins and their coordinate expression.