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

积累在植物组织表皮层的黄酮类化合物被认为在保护UV-B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，由至少7个基因组成的家族编码。在这个项目中，我们的主要目的是确定哪些基因家族成员对UV-B暴露有反应。我们开发了一个实验系统来区分基因家族的个体成员，发现7个基因中有5个基因的表达或多或少受到UV-B暴露的控制。作为了解这些基因启动子性质的第一步，从而找到改善其功能的方法，我们利用启动子/报告子结构分析了启动子活性。结果表明，基因1启动子在大豆幼苗下胚轴组织的表皮细胞中起作用，而在根组织中不起作用。除了控制启动子的物理结构外，改善基因表达的另一种方法可能是改变反式中调节基因表达的分子机制。介导CHS基因表达的转录因子值得研究。特别是在玉米中，myb同源P元件被报道通过识别启动子上的CC (T/A) ACC的核苷酸序列而激活参与类黄酮生物合成途径的反式基因。该序列也被鉴定为H-box，并且在响应UV-B照射的大豆CHS基因的每个启动子中都发现了该序列。为了寻找大豆中myb同源元素，我们进行了一些PCR实验。结果，我们鉴定出16个独立的PCR片段，其核苷酸序列与已从不同植物物种中鉴定出的myb同源基因相似度为60-80%。有些片段似乎来自自身受UV-B照射控制的基因。

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.