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
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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