1.9.16
CLONING OF DIFFERENTIALLY EXPRESSED NOVEL RICE GENES INDUCED BY MAGNAPORTHE GRISEA

ZUHUA HE, HAITAO DONG, SHIJUN CHENG, YULIANG WU, JIXIN DONG and DEBAO LI

1Biotechnologu Institute, Zhejiang Agricultural University, Hangzhou 310029, China

Background and objectives
We have applied the differential display (DD) of mRNA method to detect and clone differential expressed transcripts between rice NILs (near-isogenic lines) H7R (resistant) and H7S (susceptible) which were challenged with Magnaporthe grisea.The cDNA gene tags obtained from DD analysis were reamplified by PCR and screened by Southern and Northern blot analysis.Two sequenced tags, named RMa1 and Rma3, were checked with GeneBank and proved to be novel genes induced by M. grisea.

Materials and methods
A pair of rice NILs, H7R and H7S, were used. H7R contained gene pi-hl(t) which is resistant to race ZB15 of M. grisea [1]. Rice seedlings were inoculated with M. grisea ZB15. Total RNA was isolated after 8-24 of inoculation. The DD technique was employed as described by Peng and Pardee with some modification [2]: 120 combinations of anchor and arbitrary primers were investigated.The differentially displayed products were re-amplified and hybridized with labelled cDNA probes from either of the induced H7R and H7S rice lines, respectively, followed with Northern blotting. Selected positive fragments were inserted into the pGEM-T vector and sequenced.

Results and conclusions
In total, 48 differential cDNA fragments were obtained from H7R and H7S, which were induced by M. grisea. These fragments could be classed into three groups: (i) those only appearing or disappearing in H7R; (ii) those only appearing or disappearing in H7S; and (iii) those appearing or disappearing in H7R and H7S. Among the 48 fragments, two cDNA clones, named Rma1 and Rma3, were confirmed by Southern and Northern blotting. Rma1 appeared in both H7R and H7S with 381 base pairs in length, it has a poly(A) tail, a termination codon and poly(A) signal. There are 48 bases at the 3' end which share the same sequence as the human tumor suppressor gene SNC19. Rma3 is 198 bases in length and shares high homology with Serratia marcescens C protein. We propose that Rma1 and Rma3 are candidate defence related genes.

References
1. He Zuhua, Shen Zhontan, Huang Danian et al., 1992. Acta Agriculturae Zhejiangensis 18, 109 (in Chinese).
2. Peng l, Pardee AB,1992. Science 257, 967.