【作者】 闫海芳；

【导师】 沈海龙； 李玉花；

【作者基本信息】 东北林业大学 ， 森林培育， 2003， 硕士

【摘要】 本研究是以光敏感型津田芜菁和光不敏感型赤丸芜菁为试材，利用Northern杂交技术，对花青素生物合成途径中与花青素合成相关的主要结构基因PAL、CHS、CHI和AS及光信号传递相关的基因PHYA、COP1、CIP7和HY5的表达进行了检测。Northern分析采用的RNA样品，是从上述两品种的光处理和暗处理的肉质根部周皮层中提取的总RNA。采用的探针中，PHYA、COP1、CIP7、HY5及CHS基因的探针是利用芜菁的肉质根周皮层的总RNA合成，PAL和CHI基因的探针是利用草原龙胆花瓣的总RNA合成，AS基因的探针是利用草莓果实的总RNA合成。另外，利用HPLC液相分析系统，对两品种的光处理和暗处理样品中花青素和主要糖分(果糖、葡萄糖、蔗糖和麦芽糖)的含量进行了检测分析。对与花青素积累有明显相关的CHS基因进行了克隆。 通过实验得到如下结论： 1，光不敏感型的赤丸芜菁肉质根周皮中，光处理和暗处理都有大量花青素合成。而光敏感型的津田芜菁肉质根周皮中，只有光处理有大量花青素合成，暗处理几乎无花青素合成。 2，两品种肉质根及叶片等各组织的中葡萄糖的含量较果糖、蔗糖和麦芽糖的含量高，但葡萄糖的积累与花青素形成之间无明显相关性。两品种试材的肉质根肉质部分中四种糖的总含量高于其它部分，而叶片中含量极少。津田芜菁周皮中的果糖含量，光处理明显高于暗处理，且随光照处理时间的延长果糖含量逐渐提高，并以较高的含量稳定下来；其它三种糖含量光处理前后无明显变化。 3，CHS基因的表达，在光敏感型津田芜菁的光处理与暗处理之间有明显的差异；对津田芜菁地下根暗处理部分再进行光照处理，随光照处理的时间延长，CHS基因的表达量也逐渐提高，说明CHS基因的表达受光诱导，且表达量与花青素的积累呈正相关。在光不敏感型赤丸芜菁的不同光处理的试材中CHS基因的表达无明显的差异。因此，我们认为在光敏感型试材中CHS基因的表达对花青素的积累起关键的作用。AS基因的表达量与花青素的合成量相关，且在光敏感型津田芜菁中，AS基因的表达受光诱导。而光信号传递因子中PHYA的表达在两种品种试材中存在差异，只有光敏感型津田芜菁暗处理的试材中有表达。COP1和HY5基因在两种品种试材中，光处理与暗处理都表达，暗处理表达量比光处理少。CIP7的表达量则在两种品种试材间及光处理与暗处理中都无明显差异。更多还原

【Abstract】 Two cultivars of Brassica campestris L. ssp. rapifera, light-independent ’Akamaru’ turnip and light-dependent ’Tsuda’ turnip, were used as test material in this experiment. The expression patterns of PAL, CHS, CHI, AS, PHYA, COP1, CIP7 and HY5 were analyzed by Northern Hybridization. PAL, CHS, CHI and AS are the principal structural genes correlated with anthocyanin biosynthesis in anthocyanin biosynthesis pathway; PHYA, COP1, CIP7 and HY5 are the genes correlated with light signal transmission in anthocyanin biosynthesis pathway. Total RNA samples used in Northern blotting were extracted from the light-exposed and light-interrupted cortex tissue of tuberous root of the two turnip cultivars. The probes of CHS, PHYA, COP1, CIP7 and HY5 genes were synthesized by total RNA extracted from the cortex tissue of tuberous root of turnip, the probes of PAL and CHI were synthesized by total RNA extracted from the petal tissue of Eustoma, and the probes of AS were synthesized by total RNA extracted from the fruit tissue of strawberry. The content of anthocyanin and main sugars (fructose, glucose, sucrose and maltose) in the light-exposed and light-interrupted samples of the two turnip cultivars were analyzed by HPLC. The CHS gene which is obvious correlated with anthocyanin accumulation was cloned as well.The results are as follows:1. A large quantity of anthocyanin was synthesized in both of the light-exposed and light-interrupted cortex tissue of tuberous root of light-independent ’Akamaru’ turnip, but only in the light-exposed cortex tissue of tuberous root of light-dependent ’Tsuda’ turnip. There was almost no anthocyanin synthesized in light-interrupted cortex tissue of tuberous root of light-independent ’Tsuda’ turnip.2. Glucose’ s content was more abundant than that of fructose, sucrose and maltose in tuberous root and leaf tissues of the two cultivars, but there were no obvious correlations between glucose accumulation and anthocyanin biosynthesis. The content of the four sugars in tuberous tissue was higher than that in other tissues of tuberous root of the two cultivars. There were little sugars in leaf tissues. The content of fructose in the light-exposed cortex tissue of tuberous root of ’Tsuda’ turnip was obviously higher than that in light-interrupted cortex tissue. The content of fructose wasincreased gradually along with the prolongation of light-exposing time and stabled at high content level. There were no evident changes for the content of other three sugars after light-exposing.3. There was distinct difference for the expression patterns of CHS gene in light-exposed and light-interrupted tissues of light-dependent ’Tsuda’ turnip. Light-exposing the tuberous root of ’Tsuda’ turnip grown under dark condition, the expression quantity of CHS gene was gradually increased along with the prolongation of light-exposing time. This indicated that the expression of CHS gene was induced by light, and the expression quantity was positively correlated with accumulation of anthocyanin. There was no distinct difference for the expression patterns of CHS gene in light-exposed and light-interrupted tissues of light-independent ’Akamaru’ turnip. Therefore, we think that the expression of CHS gene played a key role in accumulation of anthocyanin in light-dependent materials. The expression quantity of AS gene was correlated with synthesis quantity of anthocyanin. The expression of AS gene was induced by light in light-dependent ’Tsuda’ turnip. The expression of light signal transmission factor PHYA is different in the two cultivars. PHYA gene expressed only in light-interrupted part of light-dependent ’Tsuda’ turnip. In the two cultivars, COP1 and HY5 expressed both in the light-exposed and light-interrupted parts of the two cultivars, but the expression quantity in light-exposed parts was higher than that in light-interrupted parts. The expression of CIP7 had no obvious difference in light-exposed and light-interrupted parts of two cultivars.更多还原