Analysis of rice genes induced by striped stemborer (Chilo suppressalis) attack identified a promoter fragment highly specifically responsive to insect feeding.
The objective of this study was to identify rice gene promoters that are specifically induced by feeding of the striped stemborer (Chilo suppressalis). Two PCR-selected cDNA subtractive libraries were constructed from the rice variety Minghui 63. Up- and down-regulated cDNAs induced by C. suppressalis feeding were arrayed on nylon membranes. After array hybridization and Northern blot analysis, a cDNA (B1-A04) encoding a putative subtilisin/chymotrypsin inhibitor was found to be rapidly and highly induced by C. suppressalis feeding, compared with mechanical wounding. The putative promoter region, spanning from -1,569 to +446 relative to the transcriptional initiation site was isolated, fused to the GUS gene (beta-glucuronidase reporter gene) and introduced by Agrobacterium-mediated transformation to rice. In non-infested plants, the GUS activity driven by this promoter fragment was detected in culms and panicles, but not in leaves and sheaths. At 6 h after insect feeding, GUS activity was significantly induced in sheaths and culms, but not in leaves. GUS activity and native B1-A04 gene were not induced by JA and ABA treatment. A serial deletion analysis revealed two regions (-1,569 to -1,166 and -1,166 to -582) that negatively regulate the gene expression in sheaths of non-infested plants but not in insect-infested plants. An electrophoretic mobility shift assay (EMSA) identified 7 DNA fragments with various binding activities with nuclear proteins from mechanically wounded, insect-infested and untreated plants, and their possible roles in gene regulation were speculated. This promoter fragment should have utility in development of insect resistant transgenic crops.
Hua
Hongxia
H
National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, China.
Lu
Qing
Q
Cai
Meng
M
Xu
Caiguo
C
Zhou
Dao-Xiu
DX
Li
Xianghua
X
Zhang
Qifa
Q
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D017931:DNA Primers; D018076:DNA, Complementary; D005247:Feeding Behavior; D017343:Genes, Plant; D007313:Insecta / Q000502:physiology*; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics* / Q000469:parasitology; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D017931:DNA Primers; D018076:DNA, Complementary