MILAN, ITALY
C.N.R. - Istituto Biosintesi Vegetali
Transient expression of fragments from the 5' flanking region of a zein
gene in electroporated protoplasts
--G. Giovinazzo, I. Coraggio, A. Viotti and L. A. Manzocchi
The analysis of cloned zein genes (Brown et al., Eur. J. Cell. Biol. 42:161,
1986; Boronat et al., Plant Sci. 47:95, 1986) has led to the identification
of a unique promoter arrangement, containing two promoters (zP1 and zP2)
lying approximately 1000 bases apart, and, between them, a group of short
highly conserved sequences which may play a role in the control of the
highly regulated zein expression; some of them have been recognized as
specific binding sites for nuclear proteins (Maier et al., Mol. Gen. Genet.
212:241, 1988). In the aim to identify specific regulatory sequences, we
have inserted in chimaeric plasmids, upstream from the reporter gene GUS
(b-glucuronidase: Jefferson
et al., EMBO J. 6:3901, 1987), the first 1415bp of the 5' flanking region
of the genomic zein clone E19. Fragments from this region have been inserted
in similar constructs: fragment 1 (290bp) comprises the zein transcription
start point and the zP2 promoter; fragment 2 (125bp) contains the so called
"-300" box (Maier et al., MNL 61:55, 1987); fragment 3 comprises the zP1
promoter. The plasmids were inserted by electroporation (1500 V/cm; 100msec;
square wave) in protoplasts obtained from leaves of N. tabacum SR1,
from long term suspension cultures of Black Mexican Sweet maize cells (of
scutellar origin) and from a recently established suspension culture of
A69Y maize endosperm cells. The promoter activity of the fragments has
been monitored as transient expression of the enzyme coded by the reporter
gene (Jefferson et al., EMBO J. 6:3901, 1987); the usual (Fromm et al.,
Nature 319:791, 1986) and a modification of the 35S CaMV promoter (as suggested
by Pierce et al., in Plant Gene Systems and Their Biology, p. 301, 1987)
have been used as constitutive promoter controls.
The expression of GUS activity (as pmoles 4-MU formed / hour / 105 protoplasts)
is summarized in the following table:
| PROTOPLASTS FROM: |
SR1 mesophyll |
BMS scut.cells |
A69Y endosp.cells |
| PROMOTERS |
|
|
|
| pCaGUS |
382 |
- |
- |
| DP33GUS |
- |
94 |
4032 |
| 1,2,3 |
74 |
4 |
0 |
| 1,2 (A) |
48 |
0.8 |
0 |
| 1,2 (B) |
26 |
0 |
0 |
| 1 (A) |
n.d. |
10 |
0 |
| 3 (A) |
7 |
32 |
49 |
| 3 (B) |
0 |
12 |
0 |
A69Y endosperm cultured cells were used with the aim to employ a homologous
maize protoplast system. Endosperm cultures have been shown to maintain
some tissue-specific features (Sarawitz and Boyer, TAG 73:489, 1987; Felker,
Am. J. Bot. 74:1912, 1987) and zein synthesis (although at reduced levels)
has been detected in our endosperm cell cultures (Manzocchi et al., Plant
Cell Rep. 1989, in press).
From our preliminary data we can observe that the entire zein promoter
region drives the expression of the reporter gene in a heterologous system
such as tobacco mesophyll protoplasts, while it appears inactive in both
types of maize cultured cells. Promoter activity, on the contrary, is observed
in maize cells for fragment 3, which is not active in tobacco protoplasts.
A more detailed analysis of zein transcription in endosperm cultured cells
will enable us to interpret the unexpected failure of activity of the entire
zein promoter region, and the functional activity of fragment 3.
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