Harvest -inducible genes and promoters in alfalfa.

摘要

A major limitation an the expression of same foreign proteins in transgenic plants is the toxic effect of such proteins on the host plant resulting in inhibition of normal growth and development. A solution to this problem is to control the expression of genes for such proteins by means of inducible promoters, as is frequently done in microbial systems.;The promoters of these three hi genes were fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants, GUS gene expression was observed in harvested tissue, but not in the non-harvested control tissue. The degree of blue coloration was greatest in plants containing the hi7-GUS construct and equal to or greater than plants containing the 35S-GUS construct. The hi7-GUS construct was also inducible by ABA in plants and in cultured cells. The hi promoters could potentially be utilized in a wide range of applications to produce pharmaceuticals in plants, enhance utilization of plant-based feeds, and facilitate processing of novel biomaterials.;In an attempt to develop an inducible expression system for potentially problematic proteins in plants, studies on harvest-induced gene expression in alfalfa were initiated for this thesis. Subtractive hybridization between cDNA from harvested alfalfa tissue (tester) and non-harvested tissue (driver) produced five unique cDNA clones designated as hi1, hi7, hi8, hi11, and hi12. Full-length cDNAs of hi7 and hi11 were obtained by 5'-RACE and 3'RACE (rapid amplification of cDNA ends). In addition, genomic walking was performed to isolate the 5' flanking region of hi7, hi11 and hi12. The expression patterns of hi7, hi11 and hi12 in alfalfa in response to harvesting, heat shock, and wounding were determined by northern blot analysis. The hi7 gene was induced strongly by harvesting, but not by heat shock, and wounding. In contrast, hi11 and hi12 were induced not only by harvesting, but also by other stress stimuli.