苦荞查尔酮合酶基因FtCHS1启动子的克隆及分析

采用染色体步移技术,从苦荞（Fagopyrum tataricum Gaertn.）中克隆获得FtCHS1基因5'端侧翼序列,共1038 bp,将其命名为P_FtCHS1。生物信息学分析表明,P_FtCHS1中A/T碱基含量为63.5%,含有4个可能的转录起始位点,分别位于起始密码子上游-684~-734、-692~-742、-920~-970、-929~-979 bp处,该序列包含TATA-Box和CAAT-Box等启动子核心元件以及与光、低温和激素应答等相关的功能元件。本研究进一步构建了P_FtCHS1-pBI101植物表达载体,并瞬时转化拟南芥（Arabidopsis thaliana L.）叶片,结果显示该序列可驱动GUS报告基因的表达。低温（4℃）和光照（UV-B）处理苦荞幼芽后,采用荧光定量PCR技术分析FtCHS1基因的表达量,结果表明P_FtCHS1可响应低温和紫外环境胁迫,从而引起FtCHS1基因表达量发生变化。     

Functional architecture of the light-responsive chalcone synthase promoter from parsley.

We have combined in vivo genomic footprinting and light-induced transient expression of chalcone synthase promoter derivatives in parsley protoplasts to identify cis sequences regulating light activation. The parsley chalcone synthase promoter contains two cis "units" that are light-responsive. Each unit is composed of short DNA stretches of approximately 50 base pairs, and each contains two in vivo footprints. One of the footprints in each unit covers a sequence that is highly conserved among other light- and stress-regulated plant genes. The other footprinted sequences in each unit are not related to each other. The TATA distal light-responsive unit is inherently weak but can compensate partially for the loss of the stronger TATA proximal unit. Levels of light-induced expression from either can be influenced by the presence of a region of approximately 100 base pairs located upstream of the TATA distal light-responsive unit. Combination of the light-responsive units and upstream region generates a synergistic response to light. We speculate that functional compensation generated by nonidentical, but sequence-related, cis units foreshadows combinatorial diversity of cognate trans factors.     

Developmentally specific effects of the DNA cross-linking agent mitomycin C on phosphoenolpyruvate carboxykinase gene expression in vivo: Correlation with changes in chromatin structure within the promoter region of the gene

Previous studies in our laboratory have demonstrated that genotoxic chemical carcinogens have strong preferential effects on expression of certain inducible genes at nonovertly toxic doses in vivo. The effects of the DNA cross-linking agent and chemotherapy drug, mitomycin C (MMC), on expression of the developmental and hormone-regulated gene, phosphoenolpyruvate carboxykinase (PEPCK), were examined in chick embryo liver in vivo as a function of development and were compared with changes in the chromatin structure of the PEPCK gene promoter. The liver PEPCK gene was fully hormone inducible as early as 8 days of embryonic development but was refractory to MMC until after day 10. This onset of responsiveness to MMC was correlated with qualitative changes in the pattern of DNase I hypersensitive sites (DHS) within the PEPCK promoter. There was also a gradual decrease and then a complete loss of both hormone inducibility and MMC responsiveness between 14 and 17 days of development that was correlated with a quantitative change in the overall DNase sensitivity of the liver PEPCK gene promoter over this period. These results suggest that carcinogen sensitivity of the PEPCK gene is related to its ability to respond to its normal induction signals and that chromatin structure may play a central role in these effects. © 1998 John Wiley & Sons, Inc. J Biochem Mol Toxicol 12: 325–337, 1998     

Signal transduction in the photocontrol of chalcone synthase gene expression*

The photocontrol of chalcone synthase gene expression was studied by means of promoter analyses, in vitro systems, photoreceptor mutants and microinjections, and pharmacological approaches. A 52 bp element of the promoter is necessary and sufficient to transfer light regulation. Chalcone synthase expression is primarily under the control of phyA and blue/UV photo receptors; the latter are functional even in the absence of phyA and phyB. Phytochromes seem to be soluble proteins and, within seconds of irradiation, light-dependent phosphorrylations were observed in membrane-depleted cytosol preparations, indicating very early processes of signal transduction. Microinjection and pharmacological experiments reveal that, in the phyA pathway, heterotrimeric G-proteins, cGMP and a genistein-sensitive protein kinase are involved, whereas the UV pathway includes several trimeric G-proteins and Ca/calmodulin-dependent steps.     

Changes in in vivo DNA-protein interactions in pea phenylalanine ammonia-lyase and chalcone synthase gene promoter induced by fungal signal molecules

Elicitor-induced protein bindings were detected on the several particular sequence motifs in two members of the pea PAL and CHS gene promoters by in vivo footprinting analyses. However, elicitor-induced changes rapidly reverted back to the uninduced pattern after the subsequent application of suppressor from M. pinodes.     

Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco.

Regulatory properties of a 1.4-kilobase promoter fragment of the bean chalcone synthase CHS8 gene were examined by analysis of glucuronidase (GUS) activity in transgenic tobacco containing a CHS8-GUS gene fusion. The promoter was highly active in the root apical meristem and in petals, exclusively in those cells of the inner epidermis that accumulate anthocyanins. The gene fusion was only weakly expressed in other floral organs, mature leaves, and stems. The early stages of seedling development were characterized by an apparent wound induction of the promoter in the endosperm and strong expression in the immature root, which became localized to the apical meristem and perivascular tissue at the root-hypocotyl junction. The promoter became active during lateral root formation in both the new root and damaged tissue of the main root. The gene fusion was also expressed in greening cotyledons and primary leaves but not in the shoot apical meristem. Light modulated expression in the cotyledons and root-shoot junction but had no effect on other aspects of the developmental program. Wounding or fungal elicitor treatment of mature leaves activated the promoter in a well-defined zone adjacent to the stress site. Stress induction occurred in mesophyll and vascular tissues as well as in the epidermis. We conclude that the CHS8 promoter contains cis-elements required to establish temporal and spatial control of flavonoid biosynthesis during development and in response to diverse environmental stimuli.     

化州柚查尔酮合成酶基因克隆与序列分析

利用CTAB-LiCl法提取高质量的化州柚总RNA,采用RT-PCR技术克隆查尔酮合成酶基因,获得广东道地药材化橘红资源化州柚的查尔酮合成酶基因。该基因编码区全长1176bp,编码391个氨基酸残基,与同样来源于柑橘属的查尔酮合成酶基因同源性高达98%。CTAB-LiCl法能提取高质量的化州柚总RNA,可以用于后续基因克隆和分析;克隆获得的查尔酮合成酶具有编码区,与同属植物相同基因具有高度序列同源性。