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高等植物几丁质酶基因受病害侵染、真菌激发子、乙烯、机械创伤等因素诱导转录。我们已经测定过水稻(Oryza sativa L.cv.IR 36)Ⅰ类几丁质酶基因RCH 8的DNA序列。为了研究水稻中与防卫反应有关的几丁质酶基因启动子的活性、功能和表达调控,我们以RCH 8几丁质酶基因保守的编码区序列为探针,分别在创伤处理2—24h后提取水稻(Oryza sativa L.cv.IR 36)叶片的总RNA作Northern杂交,发现创伤处理6h可以检测到几丁质酶基因的表达,12h表达水平最高。合成了一个与RCH8翻译起始密码附近反义链DNA序列互补的21-mer的寡核苷酸引物(5’-GCTCTCA-TGGTGGCAATGCAA-3’)作引物延伸实验,表明RCH8基因的转录受创伤诱导,RCH8转录起始位点位于翻译起始位点上游第42碱基A。在pUC 19载体质粒中构建了一组5’端不同程度缺失的RCH8基因启动子与GUS报告基因翻译融合的重组质粒,用PEG法转入烟草原生质体中,通过测定GUS酶活性发现RCH8启动子-1016-676之间的DNA序列缺失后表达活力显著下降,缺失到-68碱基处的启动子仅有很低的活力。
Higher plants chitinase gene by the disease infection, fungal elicitor, ethylene, mechanical trauma and other factors induced transcription. We have determined the DNA sequence of the class I chitinase gene RCH 8 in Oryza sativa L.cv.IR 36. In order to study the activity, function and expression regulation of the chitinase gene promoter involved in defense response in rice, we used the conserved coding region of RCH 8 chitinase gene as a probe, The total RNA of the leaves of Oryza sativa L.cv.IR 36 was extracted for Northern blotting. It was found that chitinase gene expression was detected 6h after wounding and reached its peak at 12h. A 21-mer oligonucleotide primer (5’-GCTCTCA-TGGTGGCAATGCAA-3 ’) complementary to the antisense strand DNA sequence near the translation initiation codon of RCH8 was synthesized as a primer extension experiment, indicating that transcription of the RCH8 gene was induced by trauma , The RCH8 transcriptional start site is located at the 42nd base A upstream of the translation initiation site. A set of recombinant plasmids with different degrees of 5’-end deletion of RCH8 gene promoter and GUS reporter gene fusion were constructed in pUC 19 vector plasmid and transformed into tobacco protoplast by PEG method. The results of GUS enzyme activity assay showed that RCH8 promoter The expression of -106-676 DNA sequence was significantly decreased after the loss of activity, the deletion of -68 base promoter only a very low activity.