The structure of pea legumin genes and their expression in transgenic tobacco

One of the characteristics of seed development is the synthesis and accumulation of large amounts of protein. In legumes, seed storage proteins are mostly salt-soluble globulins, which have been classified as either vicilins or legumins depending upon their molecular size and structure. Both classes of seed globulin have been thoroughly studied and therefore there is a large body of information about the proteins, as well as the genes which encode them. The fact that seed storage proteins are accumulated in a tissue-specific and temporally regulated manner during embryogenesis, has made them a useful model for investigations into the developmental regulation of plant gene expression at the molecular level. In this thesis, I report the results of my studies on the structure and expression of genes which encode the legumin class of seed storage protein in pea (Pisum sativum L.). Legumin is encoded by a small family of related genes and in this work two different members (LegAl and LegA2) were isolated from a phage library containing pea genomic sequences. The structure of the genes was determined by ✓ DNA sequencing which showed that both clones included a complete legumin gene, plus 1 kb of 5’ and 0.4 kb of 3' flanking sequence. The structural genes were interrupted by three short introns, and the deduced amino acid sequence of the proteins showed them both to be entirely consistent with what is known about the legumin class of protein. The structural features of the proteins encoded by LegAl and LegA2 are discussed and the results support the view that legumins show a remarkable degree of conservation among angiosperms. DNA sequence analysis also established that the two legumin genes were highly conserved in their protein coding regions, but shared far less identity in thenflanking regions, especially as the distance away from the structural gene increased. Nevertheless, there were several short blocks of sequence conservation that were noted in the 5' flanking region of both genes which were also found in the upstream regions of legumin genes from other legume species. Such regions of sequence conservation could imply a functional role in regulating legumin gene expression in the seed. Although legumin-like proteins are accumulated in the seeds of some non­ legume species, a survey of several such genes did not show any general similarity in the flanking regions when compared to their legume counterparts. In order to assess whether the LegAl and LegA2 clones represented functional genes, they were introduced into tobacco (Nicotiana tabacum L.) using Agrobacterium-mediated transformation techniques. The results from the transformation experiments showed that both genes were functional and that their expression was regulated in tobacco in a manner that was identical to that which occurs in pea. The pea legumin genes were expressed only in the seeds of the transgenic plants, and then only at a late stage in seed development. This organspecific and temporally regulated pattern of expression is known to operate through controls exerted at the level of gene transcription. In addition, the level of legumin accumulated in the transgenic tobacco seed was reduced when the plants were grown under conditions of limited inorganic sulphur, a response which also occurs in peas where it is known to be mediated through post-transcriptional processes that affect mRNA stability. Finally, the quantitative levels of legumin accumulated in the tobacco seed were estimated in order to establish whether the transgenes were efficiently expressed in the heterologous host. Although legumin did not exceed 1% of the extractable tobacco seed protein, it was concluded that both genes were capable of retaining at least 50% of the transcriptional activity found in peas. Comparison of legumin mRNA levels in pea and tobacco during seed development supported this conclusion. Therefore, both of the isolated legumin genes not only contained all of the regulatory elements necessary to maintain the qualitative and quantitative aspects of their expression, but also that these elements were correctly interpreted in a species that is not closely related to pea. In an effort to delineate the approximate location of these regulatory elements, a series of three progressive 5' end deletions (-668, -237, and -101) were prepared from the starting LegAl clone (-1203) and each of the deleted constructs was introduced into tobacco. It was found that legumin gene expression from the -668 clone was indistinguishable from LegAl indicating that the cis-acting regulatory elements are situated downstream of this position. In contrast, the -237 deletion failed to support any measurable accumulation of legumin protein or mRNA in the transgenic seed. This result was obtained in spite of the fact that all of the conserved upstream sequence elements noted in the analysis of the gene (namely, the CAP-site, TATA box, Legumin box and GC sequence) were retained within this deleted clone.