【作者】 周波；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 发育生物学， 2007， 博士

【摘要】 光对植物花青素合成的调控，主要是通过光受体接受光信号进行信号转导，启动花青素合成的代谢途径。如红光、UV-A光、蓝光、UV-B光均可通过相应的光敏色素、隐花色素(蓝光/UV—A受体)、UV-B受体诱导拟南芥花青素的合成。但在津田芜菁(Brassica rapa‘Tsuda’)中只有UV-A光可诱导花青素的合成，而蓝光却不能诱导花青素的合成。为了研究津田芜菁UV-A特异诱导的光信号转导因子与花青素生物合成相关基因之间的关系以及UV-A特异诱导的花青素合成调节特性，本论文以花青素合成光敏感型的津田芜菁(Brassica rapa‘Tsuda’)4天苗龄幼苗及生长3个月的膨大下胚轴表皮为试材，进行了花青素合成的光诱导研究；相关基因的表达谱分析；UV-A诱导的蛋白质磷酸化研究；信号因子的克隆、表达特性分析及瞬时表达研究；花青素合成关键基因CHS的启动子克隆及分析。得到以下主要结果：1 UV-A光对津田芜菁花青素的积累有特异诱导作用津田芜菁花青素的合成受太阳光的诱导，在UV-B、UV-A、蓝光、红光、远红光以及红蓝复合光、UV-A红光复合光、UV-A远红光复合光中，诱导津田芜菁花青素合成最有效的光波为UV-A。UV-A特异诱导津田芜菁幼苗下胚轴下部以及成株膨大的下胚轴表皮花青素的积累。2 UV-A诱导津田芜菁花青素合成相关基因群的表达及蛋白质磷酸化分析cDNA微阵列分析表明津田芜菁UV-A光处理后的基因表达谱与Dark处理以及蓝光、UV-B单色光处理相比有明显差异。在UV-A特异诱导表达的基因群中，花青素合成关键基因CHS受UV-A光的上调诱导表达。初步筛选出的UV-A诱导花青素合成相关的基因有查儿酮合酶基因、黄烷酮3-羟化酶基因、谷胱甘肽S转移酶基因、细胞色素P450基因、以及bHLH转录因子、类锌指蛋白、Myb类DNA结合蛋白转录因子等。蛋白质磷酸化分析表明UV-A诱导后，细胞膜蛋白和细胞质蛋白均出现了磷酸化水平的动态变化，它们的磷酸化在UV-A处理5 min后便得以检测，磷酸化的程度在处理30-45 min左右达到高峰，接着开始下降，到120 min时几乎检测不到。说明UV-A诱导蛋白质的磷酸化可能参与其信号转导。3津田芜菁类CRY1、COP1、HY5基因在UV-A诱导下的表达特性从津田芜菁中克隆了与拟南芥CRY1、COP1、HY5序列相似的同源基因，推测的氨基酸序列与拟南芥对应的蛋白质序列相比，相似性均达90％以上，而且CRY1序列中含有光合裂解酶和FAD-bingding结构域，COP1中含有Ring和WD-40结构域，HY5中含有bZIP结构域。在原生质体中的瞬时表达和定位表明CRY1、COP1分布在整个原生质体中，而HY5分布在细胞器(包括细胞核)区。在津田芜菁幼苗中，CRY1、COP1、HY5表达量很大，不受光的诱导。而成熟植株中COP1、HY5表达量明显减少，受UV-A的诱导，这可能与花青素生物合成相关基因的表达调控有关，虽然有证据表明CRY1不是津田芜菁UV-A特异诱导花青素信号转导的光受体，但假定存在的特异UV-A受体是否同CRY1共用信号转导的下游因子COP1、HY5还需要进一步的研究。4津田芜菁CHS的表达受UV-A特异诱导，其5’调控区存在着光反应元件津田芜菁花青素合成基因CHS5’非翻译区序列，与拟南芥CHS启动子区序列有很高的相似性，除了含有真核生物启动子结构普遍具有的保守序列CAAT box和TATA box外，还有与光诱导相关的保守序列ACE、ATCT-motif、BoxⅡ、G-Box、GATA-motif、GT1-motif、MRE、SP1和TCT-motif等。津田芜菁CHS在不同单色光以及UV-A诱导下的表达特性说明，只有UV-A诱导CHS基因大量表达，表明UV-A对津田芜菁CHS表达的特异诱导，存在着UV-A特异调控CHS表达的信号转导调控途径。更多还原

【Abstract】 Anthocyanin biosynthesis in higher plants is regulated by light. Photoreceptors receive lightand are involved in light signal transduction to stimulate the metabolism of anthocyaninaccumulation. In Arabidopsis anthocyanin biosynthesis is induced by red, UV-A, blue, and UV-B light through phytochromes, cryptochromes and UV-B receptor, respectively. In Brassicarapa ’Tsuda’, however, only UV-A induced anthocyanin biosynthesis. In this study, ’Tsuda’plants at the stage of 4 days or 3 months old were used as materials. The anthocyaninbiosynthesis character as induced by light, the analysis of gene expression profile, the cloningand expression of regulated gene, the cloning and analysis of promoter of CHS wereinvestigated. The main results were obtained as follows.1. UV-A specific induction of anthocyanin biosynthesis in ’Tsuda’ plants.Anthocyanin biosynthesis was induced by sunlight in ’Tsuda’ plants. When lights withspecific wave length were irradiated such as UV-B, UV-A, blue, red, far-red, blue plus red, UV-A plus red, and UV-A plus far-red lights, only UV-A induced anthocyanin biosynthesis. UV-Ainduced anthocyanin biosynthesis in both of hypcotyls of seedlings and swollen hypocotyls of’Tsuda’ plants.2. UV-A specific induction of gene expression profile of anthocyanin biosynthesis andprotein phosphorylation in ’Tsuda’ plants.Microarray analysis showed that gene expression profile induced by UV-A had obviousdifference from those by blue and UV-B. Among a set of many genes induced by UV-A, theexpression of CHS, which was a key gene of anthocyanin biosynthesis, showed up-regulation.Genes for anthocyanin biosynthesis, including chalcone synthase, flavanone 3-hydroxylase,glutathione S-transferase, cytochrome P450, bHLH transcription factor, zinc finger-like protein,and Myb-like DNA binding protein were screened out by UV-A induction.Both cytoplasmic and membrane proteins were phosphorylated in response to UV-Aexposure. The phosphorylation of these cytoplasmic and membrane proteins became detectablewithin 5 minutes after UV-A exposure. The phosphorylation extent increased gradually until30-45 min after exposure, and then declined to an almost undetectable level by 120 min. Itsuggested that the phosphorylation of proteins in response to UV-A exposure might be involvedin light signal transduction.3. The expression character of putative CRY1, COP1, HY5 induced by UV-A in ’Tsuda’plants.Homolog cDNAs of CRY1, COP1, and HY5 in ’Tsuda’ were isolated and the amino acidresidues deduced from each gene showed over 90％sequence identity to that of Arabidopsis CRY1, COP1, and HY5, respectively. Photolyase and FAD-binding domains in CRY1, Ringand WD-40 domains in COP1, and bZIP domain in HY5 were separately found in putativeCRY1, COP1, and HY5 of ’Tsuda’ plants. The transient expression of CRY1, COP1, and HY5in protoplast showed that CRY1 and COP1 were distributed in the whole protoplast and HY5was in the area of nucleus. In the seedlings of ’Tsuda’plants, the level of CRY1, COP1, HY5expression was higher than that in mature plants, but they were not induced by light. In matureplants, in contrast, the expression of COP1, HY5 was induced by UV-A. It suggested that itmight be related to the expression of anthocyanin biosynthesis genes. Although CRY1 was notshown to be a photoreceptor of UV-A signal transduction in anthocyanin biosynthesis of B.rapa ’Tsuda’, the pseudo-UV-A photoreceptor and CRY1 photoreceptor were considered toshare potentially the same regulators, ie., COP 1 and HY5.4. The expression of CHS was induced specifically by UV-A and LRE was localized in its5’-untranslation regionThe sequence of CHS 5’ UTR in ’Tsuda’ plants had high identity to that of the promoter ofCHS in Arabidopsis. It contained CAAT box, TATA box and light-induced motif such as ACE,ATCT-motif, BoxⅡ, G-Box, GATA-motif, GT1-motif, MRE, SP1 and TCT-motif. Theexpression character of CHS induced by light with different wavelengths showed that only UV-A induced the expression of CHS in ’Tsuda’ plants. It may be possible that a UV-A signaltransduction way specifically induces the expression of CHS in B. rapa ’Tsuda’.更多还原