Cloning And Expression Profile Of A Herbivore Specifically Induced Rice Gene OsSABATH And Its Promoter Analysis

We previously found that infestation by rice stripped stem borer Chilo suppressalia specifically up-regulated the expression levels of a gene OsSABATH in rice, which belongs to a member of SABATH methyltransferases. In order to elucidate the biological function of the OsSABATH gene in rice and identify herbivore feeding responsive cis-acting elements, the gene OsSABATH was cloned and its expression profile was analyzed. Moreover, the OsSABATH protein was purified from E. coli expressing the OsSABATH gene and the transgenic plants with deletion mutationns of the gene promoter were obtained through using Agrobacterium-mediated transformation system,. The main results are as follows:1. A 2139 bp cDNA of the gene OsSABATH, containing an open reading frame of 990bp, was cloned. The amino acid sequence deduced from the open reading frame revealed that OsSABATH encodes a protein of 329 amino acids with a calculated molecular weight of 37.4KDa. Alignment of the protein sequence of OsSABATH with other sequences from databases revealed that OsSABATH has a high level of identity to a known OsSAMT. OsSABATH mRNA expression is high in roots, low in leaves, stems and flowers. The expression levels of the OsSABATH gene in rice were up-regulated after the plants were infested by Chilo suppressalis, Sogatella furcifera or Chaphalocrocis medinalis: the expression levels began to increase at 2 h after infestation and reached high levels at 8 h. Nilaparvata lugens infestation or mechanical wounding, however, did not increase the OsSABATH mRNA expressioa Exogeous application of SA, JA or ethylene, three kinds of signal molecules, did not enhance the expression levels of the OsSABATH gene in rice within 24 h.2. A prokaryotic expression vector pET32a(+)-OsSABATH for OsSABATH gene were construced and expressed in E.coli. SDS-PAGE analysis showed that the recombinant protein was obviously expressed at 3 h after induced by IPTG. The recombinant CteSABATH protein was purified by using the Ni-NTA protein purification column.3. A 2050bp promoter sequence upstream of this gene was amplified using a pair of primers designed on the basis of the sequence of the OsSABATH and rice genome sequence database. With the aid of PLACE program, several cis-acting elements in the promoter sequence, including W-box, DOF binding motif, GT-1 element, and BIHD1 binding motif, were recognized. For analyzing the activity of the promoter sequence, the promoter region sequence and its 5'deletion constructions were fused to the GUS reporter gene in pCAMBIA1391 vector, and by using Agrobacterium-mediated transformation system, three kinds of transgenic plants with the promoter region sequence (PRP1,2050bp) or its 5'deletion constructs (PRP2, 1360bp or PRP3,998bp), named PRP1, PRP2 and PRP3, were obtained. In PRP1 transgenic plants, Chilo suppressalis infestation significantly increased GUS activities compared to non-manipulated plants but not wounded plants. In PRP2 and PRP3 transgenic plants, Chilo suppressalis infestation significantly increased GUS activities compared to both non-manipulated and wounded plants. This suggests that wound responsive cis-elements in the secquence between PRP1 and PRP2 and herbivore responsive cis-elements in the PRP3 sequence may exist.