Transgenic plants to study regulatory sequences
Dozens of genes in plants are affected by light and many of these regulated by phytochrome. The expression of phytochrome gene is depressed and that of RUBISCO (ribulose, 1,5 bisphosphate carboxylase/ oxygenase) gene is stimulated by red light. RUBISCO is involved in CO2 fixation and is the most abundant enzyme on the earth. It consists of two subunits, each encoded by a separate gene, the smaller subunit rbcS encoded by a nuclear gene and the larger subunit rbcL encoded by a chloroplast gene. Although, rbcS from different plants are fairly similar, small differences, that exist,
Gene for chlorophyll a/b binding protein (Cab).
Examples of the transfer of Cab gene from pea, wheat and Nicotiana plumbaginifolia are listed in Table 37.4. In this case it was shown that a 247 bp sequence for pea CabAB80 (-347 to -100) and a 268 bp sequence (-357 to -89) for wheat Cab-l,
Thermal stress induces the transcription of a small set of heat shock genes in diverse organisms, and also reduces the expression of many other vital genes. The upstream region of heat shock genes regulating their expression is highly conserved, so that when a gene construct carrying nptII reporter gene, fused with upstream region of heat shock gene hsp70from Drosophila, was introduced into tobacco, induction of NPTII expression due to heat shock was comparable to that of endogenous plant heat shock genes (inducible in all organs except pollen). Similarly, in maize a homologue of above gene, hsp70 with 1.1 kilobases of upstream sequence, when introduced in transgenic petunia, exhibited heat inducibility.
Alcohol dehydrogenase gene expresses under anaerobic conditions leading to oxygen deprivation. Using transgenic plants, it could be shown that regulatory sequences upstream to ADH gene from maize lead to suppression (downregulation) of gene activity under aerobic conditions, this activity being normal and uninducible under anaerobic conditions. The inability of the ADH upstream regulatory sequences to induce gene expression became evident when these sequences failed to induce chloramphenicol acetytransferase (CAT) expression under aerobic as well as anaerobic conditions in transgenic tobacco plants. Other enhancer sequences (from octopine synthase or CaMV35S gene), when transferred at a position upstream of ADH gene, led to induction under anaerobic conditions. Therefore, under normal conditions, ADH activity is inhibited under aerobic conditions due to negative control and expression is promoted under anaerobic conditions simply due to the absence of a negative control rather than the presence of positive induction.
The genes for seed storage proteins provide an excellent example of a cell and tissue specific expression. Using transgenic plants, sequences could be identified which were responsible for tissue or cell specific expression of these genes. For instance, when wheat glutenin gene or barley hordein gene (with its regulatory sequence) was fused to chloramphenicol acetyltransferase (CAT) gene and introduced into tobacco, CAT activity was found only in the endosperm. Another reporter gene utilized for the same purpose is β-glucuronidase (GUS) gene, which also showed endosperm specific expression when fused with a wheat glutenin gene (with its regulatory sequence). The regulatory sequence thus identified was called endosperm box of the storage protein genes.
5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS) is an enzyme involved in aromatic amino acid biosynthetic pathway and is the target of glyphosate based herbicides. In petunia, enzyme is synthesized in flowers at a high level and is hardly found in leaves, although in tomato it is synthesized both in flowers and leaves. Transgenic tobacco plants carrying CAT gene or GUS gene fused with deletion mutant forms of EPSPS gene were analysed and it was concluded that sequences between -1800 to -800 of petunia EPSPS gene were responsible for tissue specific expression of this gene.