Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation
Mj. Leech et al., Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation, PLANT MOL B, 38(5), 1998, pp. 765-774
We have created a population of transgenic tobacco plants carrying cDNAs en
coding two consecutive enzymes from early stages in monoterpenoid alkaloid
biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan decarbo
xylase ((tdc)) and strictosidine synthase (str1) together with a selectable
marker gene, were introduced on a single transforming plasmid into tobacco
leaves by particle bombardment. Analysis of 150 independent transgenic pla
nts at the DNA and RNA levels demonstrated a range of integration events an
d steady-state transcript levels for the tdc and str1 transgenes. Southern
blot analysis indicated that the tdc and str1 transgenes were integrated at
least once in all 150 transformants giving a 100% co-integration frequency
of the two unselected genes carried on the same plasmid. A comparison of S
outhern and northern data suggested that in 26% of the plants, both tdc and
str1 transgenes were silenced, 41% demonstrated a preferential silencing o
f either the tdc or the str1 transgene, with the remaining 33% of the plant
s expressing both transgenes. We observed no clear correlation between the
number of integration events of a specific transgene and the levels of accu
mulated transcript. Twenty plants representing the range of molecular diver
sity in the transgenic population were selected for further analysis. Seeds
were collected from self-fertilised transformants and germinated on medium
containing kanamycin. Seedlings were harvested after 7 weeks and TDC and S
TR1 enzymatic assays were carried out. We observed a 24- and 110-fold varia
tion in levels of TDC and STR1 activities, respectively. Our data correlate
molecular diversity with biochemistry and accumulation of end-product and
provide a detailed molecular and biochemical characterization of transgenic
plants transformed with a single plasmid carrying two genes of secondary m
etabolism.