【作者】 温泉；

【导师】 吴能表；

【作者基本信息】 西南大学 ， 植物学， 2012， 硕士

【摘要】 为了阐明不同强度UV-B对黄连体内小檗碱合成代谢的初步调控机理及黄连对UV-B的生理响应。试验设置了CK(自然光)、UL(0.05 W·m-2)、UM(0.10 W-m-2)、UH (0.20 W-m-2)4个处理组,探讨长期(35d每天辐射3h) UV-B辐射下黄连初生代谢中光合特性、PPP途径和次生代谢中酪氨酸酶及黄连根、茎、叶中小檗碱含量的变化特性；同时探讨短期(1～9h) UV-B辐射对黄连SOD、SOD、CAT同工酶、逆境指标的变化特性,以及短时UV-B(0～30min)处理黄连叶片后,运用[γ-P32]ATP放射性同位素测定蛋白激酶活性变化特性。得到如下结果：1.蛋白激酶活性的激活具有瞬时性,通过液体闪烁计数器对γ-P32放射强度表明,UL、UM辐射强度处理黄连叶片1min后,蛋白激酶活性显著增强,且UL组蛋白激酶活性要大于UM组,UL强度辐射2min后,蛋白激酶活性开始下降,到30min降至最低,UM强度辐射1min后,蛋白激酶活性开始回落,5min后基本恢复到正常水平,而重度UV-B辐射下,黄连叶片内的蛋白激酶活性无明显变化。2.在UH辐射强度下,黄连叶片中光合色素、qN、Fo、ETR以及葡萄糖-6-磷酸脱氢酶活性、根中小檗碱含量均较其它组低。而处于UM辐射下的黄连,光合作用能力、葡萄糖-6-磷酸脱氢酶活性、酪氨酸酶活性以及根中小檗碱含量均高于对照组,且黄连会通过胁迫应激反应来抵御轻、中度UV-B辐射。3.UV-B辐射3h后,SOD1 (Rf=0.125)、SOD2 (Rf=0.312)、CAT1 (Rf=0.428). POD3 (Rf=0.290)、POD4 (Rf=0.636)谱带被先后诱导,随着UV-B辐射时间的增长,这些谱带开始丢失或减弱,尤其处于重度UV-B胁迫下,CAT1 (Rf=0.428)、POD3 (Rf=0.290)谱带会提前丢失；此外,UV-B辐射使得叶片中MDA、可溶性糖、脯氨酸含量显著高于对照组,但UV-B辐射7h后,除UL组脯氨酸上升外,各组MDA、可溶性糖、脯氨酸含量开始有所下降,可溶性蛋白谱带数增多。4.在本试验条件下,UL辐射强度处理35d最有利于黄连叶片中小檗碱含量的积累,处理21d有利于根中小檗碱的积累,增幅为46.9%；UM辐射强度处理黄连14d后,黄连叶中小檗碱含量的迅速积累,增幅达到72.9%,35d有利于根中小檗碱含量的积累；UH辐射强度处理黄连超过21d会导致根及叶中的小檗碱含量明显下降,但使得茎中小檗碱积累量增加。结论：UL(轻度)、UM(中度)辐射有利于诱导根和叶中小檗碱含量的积累,原因可能是黄连处于UL、UM辐射时,蛋白激酶活性升高,激活相关酶的活性及基因的表达,增强光合作用及PPP途径,以致提供较多的次生代谢物前体物及次生代谢物所必需的NADPH,进而小檗碱的含量也相应提高。且UL、UM组辐射强度会诱导黄连增加抗氧化酶同工酶的表达量及谱带数,积累可溶性糖、可溶性蛋白等抗性物质来提高自身抗性能力,虽然UH(重度)UV-B辐射会导致茎中小檗碱含量增加,但减少了根及叶中的小檗碱,这可能是重度UV-B辐射持续时间过长将使得黄连自身调节系统被破坏,代谢开始紊乱,小檗碱合成受阻。更多还原

【Abstract】 In order to elucidate the regulation mechanism of different intensity of UV-B on the metabolism of the C. chinensis body berberine and C. chinensis physiological response to UV-B. The experimental set four treatment groups:CK (natural light), UL (0.05 W·m-2), UM (0.10 W·m-2), UH (0.20 W·m-2) to explore long-term (35d daily radiation 3h) UV-B radiation C. chinensis primary metabolism in the photosynthetic characteristics of PPP pathways and secondary metabolites in tyrosinase activity and C. chinensis root, stem, leaf content of berberine change characteristics; to explore short-term (1-9h) UV-B in characteristics of POD, SOD, CAT, isozyme, Stress Indexes of C. chinensis, as well as using UV-B (0-30min) to deal with C. chinensis leaves, the use of [y-P32] ATP isotope is to test activity characteristic of protein kinase. Results are as follows:1. Kinase activity activated transiently, the radiation intensity of y-P32 by liquid scintillation counter. UL, UM radiation processing C. chinensis leaves for I min, the kinase activity was significantly enhanced, and the UL group kinase activity is greater than the UM group, the kinase activity began to decline after the intensity of UL radiation for 2 min, and to a minimum level after 30 min, the UM intensity radiation 1min after the kinase activity began to fall, basically returned to normal levels after 5 min. However, kinase activity of C. chinensis leaves stay balance under severe UV-B radiation.2. Photosynthetic pigments in the C. chinensis leaves, qN, the Fo ETR and the glucose-6-phosphate dehydrogenase activity, root of berberine content is lower than other groups under UH radiation condition. However, photosynthetic capacity, glucose-6-phosphate dehydrogenase activity, tyrosinase activity and the content of berberine in roots were higher than the control groups under UM radiation condition, the phenomenon may be due to the fact that C. chinensis may produce stress response against the light and moderate UV-B radiation.3. UV-B radiation after 3h, the SOD1 (Rf=0.125), the SOD2 (Rf=0.312), CAT1 (Rf= 0.428), POD3 (Rf=0.290), POD4 (Rf=0.636) band was successively induced, with the time the growth of UV-B radiation, and these bands began to be lost or weakened, especially in severe UV-B stress, CAT1 (Rf=0.428), POD3 (Rf=0.290) bands will be lost in advance; In addition, UV-B radiation makes the leaves of MDA, soluble sugar, proline content was significantly higher than the control, UV-B radiation 7h, in addition to the UL group of proline increased in each group of MDA, soluble sugar, proline content began to decline in the number of soluble protein bands increased.4. Accumulation of Berberine in C. chinensis leaves happens under UL radiation processing 35d condition, moreover, handle 21 d is conducive to the accumulation of berberine in the root, with an increase of 46.9%; the UM radiation treatment C. chinensis 14d, Berberine Content of the leaves of C. chinensis with the rapid accumulation, increase comes up to 72.9%,35d there is conducive to the accumulation of the content of berberine in the root; UH radiation processing C. chinensis more than 21d will lead to decreased berberine content in the roots and leaves, but the content of berberine in the stem show the increase trend.Conclusion:UL (mild), UM (moderate) radiation are beneficial to induce the accumulation of berberine content in roots and leaves, probably due to C. chinensis in the UL, the UM radiation, the increased kinase activity, activation of the related enzyme activity and gene expression, enhanced photosynthesis and PPP pathway, resulting in more secondary metabolites precursors and secondary metabolites required NADPH, and then the content of berberine also increased accordingly. Furthermore, the radiation intensity induced C. chinensis increased antioxidant isoenzyme expression and the number of bands, resistance to the accumulation of soluble sugar, soluble protein substances to improve their resistance ability in UL, UM group. Although the UH (severe) of UV-B radiation can make the content of berberine accumulated in the stem, the content of berberine of the root and leaves of C. chinensis reduced, the reason probably is that severe UV-B radiation long duration will destroy its own regulate conditioning systems, furthermore, metabolism began to disorder, berberine synthesis blocked.更多还原