Wu Xue-Feng, Wang Chun-Lian, Xie En-Bei, Gao Ying, Fan Ying-Lun, Liu Pi-Qing, Zhao Kai-Jun
National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), Key Laboratory of Crop Genetics and Breeding, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
Planta. 2009 May;229(6):1231-42. doi: 10.1007/s00425-009-0911-9. Epub 2009 Mar 11.
We have previously isolated a Brassica juncea cDNA encoding a novel chitinase BjCHI1 with two chitin-binding domains (Zhao and Chye in Plant Mol Biol 40:1009-1018, 1999). The expression of BjCHI1 was highly inducible by methyl jasmonate (MeJA) treatment, wounding, caterpillar feeding, and pathogenic fungal infection. These observations suggest that the promoter of BjCHI1 gene might contain specific cis-acting elements for stress responses. Here, we report the cloning and characterization of the BjCHI1 promoter. A 1,098 bp BjCHI1 genomic DNA fragment upstream of the ATG start codon was isolated by PCR walking and various constructs were made by fusing the BjCHI1 promoter or its derivatives to beta-glucuronidase reporter gene. The transgenic Arabidopsis plants showed that the BjCHI1 promoter responded to wounding and MeJA treatment, and to treatments with either NaCl or polyethyleneglycol (PEG 6000), indicating that the BjCHI1 promoter responses to both biotic and abiotic stresses. A transient gene expression system of Nicotiana benthamiana leaves was adopted for promoter deletion analysis, and the results showed that a 76 bp region from -695 to -620 in the BjCHI1 promoter was necessary for MeJA-responsive expression. Furthermore, removal of a conserved T/G-box (AACGTG) at -353 to -348 of the promoter greatly reduced the induction by MeJA. This is the first T/G-box element identified in a chitinase gene promoter. Gain-of-function analysis demonstrated that the cis-acting element present in the 76 bp region requires coupling with the T/G-box to confer full magnitude of BjCHI1 induction by MeJA.
我们之前已分离出一个芥菜型油菜cDNA，其编码一种具有两个几丁质结合结构域的新型几丁质酶BjCHI1（Zhao和Chye，《植物分子生物学》，第40卷，第1009 - 1018页，1999年）。BjCHI1的表达可被茉莉酸甲酯（MeJA）处理、创伤、毛虫取食以及病原真菌感染高度诱导。这些观察结果表明，BjCHI1基因的启动子可能包含用于应激反应的特定顺式作用元件。在此，我们报告BjCHI1启动子的克隆与特征分析。通过PCR步移法分离出ATG起始密码子上游1098 bp的BjCHI1基因组DNA片段，并通过将BjCHI1启动子或其衍生物与β - 葡萄糖醛酸酶报告基因融合构建了各种载体。转基因拟南芥植株表明，BjCHI1启动子对创伤和MeJA处理以及NaCl或聚乙二醇（PEG 6000）处理均有反应，这表明BjCHI1启动子对生物和非生物胁迫均有反应。采用本氏烟草叶片的瞬时基因表达系统进行启动子缺失分析，结果表明BjCHI1启动子中从 - 695至 - 620的76 bp区域对于MeJA响应性表达是必需的。此外，如果去除启动子 - 353至 - 348处保守的T/G - 盒（AACGTG），则MeJA诱导作用会大大降低。这是在几丁质酶基因启动子中鉴定出的首个T/G - 盒元件。功能获得分析表明，76 bp区域中存在的顺式作用元件需要与T/G - 盒结合，才能赋予MeJA对BjCHI1的完全诱导作用。
