1.2.17
EXPRESSION ANALYSIS OF A GENE FAMILY IN FRENCH BEAN (PHASEOLUS VULGARIS) INDUCED BY CARBOHYDRATE ELICITOR

E FUKUSAKI, Ta WATANABE, Te WATANABE, S KAJIYAMA and A KOBAYASHI

Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan

Background and objectives
Plants respond actively to infection and other environmental stresses by synthesizing phytoalexin and defence-related proteins. This response is known to be induced by compounds known as elicitors. These are often oligosaccharide fragments dereived from chitin, chitosan, beta-glucan and pectic acid. In most of the elicitor studies, crude elicitor fractions have been used because of the difficulty in preparing pure elicitors. We have developed convenient procedures to obtain pure biotic elicitors to legumes from naturally occurring polysacchaarides and reported novel elicitors such as oligo-beta-glucan fragments [1, 2] and partially de-acetylated oligochitin fragments [3, 4]. We have employed an elicitor assay system based on detection of phytoalexin accumulation in legume plants. Such a method has been widely used for elicitor studies, however it may not be appropriate for high throughput screening of elicitor. Therefore, it is essential to develop a simple and sensitive elicitor assay system for further elicitor studies. We have focused on specific gene expressions induced by elicitors and tried to establish a new elicitor assay. We have performed mRNA differential display to obtain appropriate genes as indexes for gene expression-based elicitor assay. We present the results of expression analysis of a gene family in French bean (Phaseolus vulgaris) induced by a carbohydrate elicitor, laminaran.

Materials and methods
10-day-old P. vulgaris leaves were treated with laminaran elicitor (1000 p.p.m.). Total RNA was extracted from an elicitor-treated leaf and a non-treated leaf, separately. mRNA differential display was performed according to Yoshida's method [5] with slight modifications. In brief, DNA contaminations in total RNA preparations were digested with DNAse I. After separation on 1.5% agarose gel, bands of interest were cut and eluted DNA fragments were inserted into the plasmid vector pCRII (TA Cloning Kit, Invitrogen, San Diego, CA). The obtained cDNAs were subjected to sequencing analysis followed homology search studies using BLAST.

Results and conclusions
As a result, six novel genes have been obtained. These showed substantial homologies to the genes described below, respectively: (i) Glycine max UDP-glucose dehydrogenase; (ii) Glycine max calnexin; (iii) Arabidopsis thaliana APK1 gene for protein tyrosine-serine-threonine kinase; (iv) Acetobacter aceti cya gene for ubiquinol oxidase; (v) Capsicum annuum mRNA for plastid fusion and/or translation factor; (vi) pea chloroplast photosystem II gene encoding the D2 and 44-kDa chlorophyll-binding protein. Northern analysis of the genes obtained is in progress.

References
1. Kobayashi A, Tai A, Kawazu K, 1995. Journal of Carbohydrate Chemistry 14, 819-832.
2. Tai A, Ohsawa E, Kawazu K, Kobayashi A, 1996. Zeitschrift fur Naturforschung 51, 15-19.
3. Akiyama K, Kawazu K, Kobayashi A, 1995. Zeitschrift fur Naturforschung 50, 391-397.
4. Akiyama K, Kawazu K, Kobayashi A, 1995. Carbohydrate Research 279, 151-160.
5. Yoshida KT, Naito S, Takeda G, 1994. Plant Cell Physiology 35, 1003-1009.