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

植物组织中积累的类黄酮类化合物被认为在UV-B防护中起重要作用。查尔酮合酶(CHS)是大豆类黄酮生物合成第一步反应的催化剂，由至少7个基因组成的家族编码。我们在这个项目中的主要目的是确定该基因家族中的哪个成员在对UV-B暴露的响应中表达。我们开发了一个实验系统来区分该基因家族的个别成员，并发现7个基因中有5个或多或少受到UV-B辐射的控制。作为了解这些基因启动子性质的第一步，从而找到改进其功能的方法，利用启动子/报告结构来分析启动子的活性。我们的结果表明，基因1启动子在大豆幼苗下胚轴组织的表皮细胞中发挥作用，而在根组织中不起作用。除了控制启动子的物理结构外，还可能通过改变调节反式基因表达的分子机制来提高基因的表达。介导CHS基因表达的转录因子被认为是值得研究的。特别是在玉米中，通过识别启动子上CC(T/A)ACC的核苷酸序列，MYB同源的P元件被报道在参与类黄酮生物合成途径的反式基因中被激活。该序列也被鉴定为H-box，存在于响应UV-B辐射的大豆CHS基因的每个启动子中。为了寻找大豆MYB同源基因，我们进行了一些聚合酶链式反应实验。结果，我们鉴定了16个独立的PCR片段，它们与已从不同植物中鉴定的MYB同源基因的核苷酸序列相似性在60%-80%之间。其中一些片段似乎来自于基因本身，这些基因本身受到UV-B辐射的控制。

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.