Molecular analysis of soybean chalcone synthase gene expression and UV-B-tolerance.

植物组织中积累的黄酮类化合物被认为在UV-B防护中起重要作用。查尔酮合成酶（Chalcone synthase，CHS）是大豆中催化类黄酮生物合成的第一步反应，由一个至少有7个基因的家族编码。我们在这个项目中的主要目的是确定哪个基因家族的成员在响应UV-B暴露时表达。我们开发了一个实验系统来区分基因家族的各个成员，发现7个基因中有5个或多或少地受到UV-B暴露的控制。作为理解这些基因的启动子的性质的第一步，以便找到改善其功能的方法，通过利用启动子/报告子构建体分析启动子活性。结果表明，CHS基因1启动子在大豆幼苗下胚轴表皮细胞中起作用，而在根组织中不起作用，除了改变启动子的物理结构外，还可能通过改变调控基因表达的分子机制来提高CHS基因的表达水平，因此，对CHS基因表达调控的转录因子的研究具有重要意义。特别是在玉米中，据报道MYB同源P元件通过识别启动子上的CC（T/A）ACC核苷酸序列而在参与类黄酮生物合成途径的转基因中激活。该序列也被鉴定为H-box，存在于所有对UV-B辐射敏感的大豆CHS基因的启动子中。我们进行了一些PCR实验，以寻找大豆MYB同源元件。其结果是，我们确定了16个独立的PCR片段，显示核苷酸序列的相似性范围为60-80%的myb同源基因已经确定从不同的植物物种。有些片段似乎来自基因，而这些基因本身是受UV-B辐射控制的。

Flavonoids which accumulate in the epidermal layrs of plant tissues are considered to play an important role in UV-B protection. Chalcone synthase (CHS), catalyzing the first step reaction of flavonoid biosynthesis, is encoded by a family of at least 7 genes in soybean. Our primary aim in this project was to identify which member of the gene family was expressed in responsive to UV-B exposure. We developed an experimental system to distinguish individual members of the gene family and found that 5 out of the 7 genes are more or less under the control of UV-B exposure for their expression. As a first step toward understanding the property of the promoters of these genes, so as to find a way to improve their function, promoter activity was analyzed by utilizing promoter/reporter constructs. Our results suggested that gene 1 promoter functioned in the epidermal cells of hypocotyl tisues but not in those of root tissues of soybean seedlings.Another way to improve expression of the genes, other than manipulating physical structures of promoters, may be found in changing the molecular mechanisms which modulate gene expression in trans. Transcription factors mediating CHS gene expression were considered worth studying. Especially, in maize, MYB-homologous P element is reported to activate in trans genes involved in the pathway of flavonoid biosynthesis by recognizing the nucleotide sequence of CC (T/A) ACC on the promoters. This sequence has also been idebtified as H-box and was found in every promoter of the soybean CHS genes responding to UV-B irradiation. We have performed some PCR experiments in search of MYB-homologous elements in soybean. As a result, we identified 16 independent PCR fragments which showed nucleotide-sequence similarities ranging 60-80% with the myb-homologous genes already identified from different plant species. Some of the fragments seemed to have derived from the genes which themselves are under the control of UV-B irradiation.