Analyzing Gene Expression With Transgenic Plants

I. Are genes expressed in transformed plants as they are in the plant from which they were isolated?

1. Transformed petunia callus expresses a pea gene. (Broglie et al. Science 224:838, 1984) This was an early experiment that showed that introduced genes are expressed in transformed cells. Not only were the genes expressed, but they responded to the same environmental stimuli as the whole plant.
2. Transformed genes are expressed in a normal developmental manner. (Sengupta-Gopalan et al. PNAS 82: 3320 1985). This experiment showed that introduced genes can be regulated in the correct developmental manner in transgenic plants.
3. Several genes can be expressed in a transgenic plant (Okamuro et al. PNAS 83: 8240 1986). Two genes were introduced into a plant and the transgenic plant correctly expressed them both.
4. Monocot genes could be expressed correctly in a dicot. (Lamppa et al. Nature 316:750 1985) The wheat genes which encode the chlorophyll a/b binding proteins where expressed in the correct tissues in transgenic tobacco plants.

II. Can genes be targeted to specific organelles?

Transit peptide - amino acid sequences on the amino-terminal portion of a protein that target the protein to a specific organelle; the peptide is removed during or immediately after transport into the organelle

Molecular and General Genetics 205:454 (1986) - rbcS transit peptide was used to incorporate an engineered protein into the chloroplast; the following construct was made;

NOS Promoter --- rbcS TP-NPT II --- NOS 3'

Result: NPT II activity was detected in the chloroplast

Nature 328:340 (1987) - the beta-subunit of ATP synthase is a nuclear-encoded peptide of this mitochondrial located protein complex; the following construct was made:

CaMV 35S Promoter --- beta subunit Transit Peptide-CAT --- 3'???

Result: CAT activity was detected in the mitochondrial fraction

III. Can differential gene expression be studied?

Phenylalanine -------> Transcinnamic acid

Products: lignin, flavanoid pigments, phytoalexins In bean, a small multigene family of 3-4 genes encode for the PAL proteins. Molecular analysis has shown differential gene expression among gene family members. Transgenic plants were developed to study the expression of one member of the family, PAL 2. The following construct was made:

PAL2 PROMOTER --- GUS --- NOS 3'

Results:

• Organ specificity (Table 1) Gene expression was highest in the stem and root and lower in the leaf. This expression mimics what is seen in the bean plant.
• Tissue distribution in the floral parts (Table 2) The highest expression was seen in the petals, the floral part that exhibits the color. The expression was lowest in the ovaries and sepals.
• Wounding (Table 1) This gene had been shown to be induced by wounding. GUS activity was also induced in the transgenic tobacco plants upon wounding.
• Light responsiveness (Table 3) The light responsiveness of this promoter was the same as that in bean. After being grown in the dark for three days, low level of expression was seen. But after transfer to light, expression dramatically increased.

1. Sequences are present that control where the gene is expressed in the plant. (Table 1,2)
2. Sequences are present that mediate the wounding response.
3. Sequences are present that mediate light responsiveness.
4. Those trans-acting factors which regulate gene expression in bean are present in tobacco.

IV. Can the effects of environmental stimuli be studied?

Patatin is a group of potato storage proteins that are represented by two different classes of proteins. Together these proteins are encoded by a multigene family. An important finding was that these genes could be expressed in the leaves of potato plants. This suggested that signals required for its expression were also located in leaves. The next question that was raised is whether certain environmental stimuli could be identified that turned on the gene expression. The logical choice was sugars since protein and sugar deposition in the developing tuber is performed in a sugar rich environment.

The following construct was made:

Class I Patatin Promoter --- GUS --- NOS 3'

Both transgenic tobacco and potato plants were obtained and analyzed for gene expression in the leaves.

Results:

• Both tobacco and potato responded dramatically to increasing leaves of sucrose in the culture medium. The level of expression differed (potato greater than tobacco), but the shape of the induction curve was similar. (Fig. 2)
• The induction of the enzyme was accompanied by an increase in the mRNA for GUS, the marker gene. (Fig. 3)
• Not all sugars could induce this expression. Fructose and glucose was able to induce the expression, individually or in concert. Only sucrose induced the high levels of gene expression.

• The class I patatin promoter contains sequences that are activated in same manner by the presence of sucrose.
• The trans-acting factors required for patatin gene expression are present in tobacco leaves.

Copyright © 1998. Phillip McClean