【作者】 林建中；

【导师】 刘选明； 林辰涛；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 博士

【摘要】 光照能够强烈的影响植物的初生代谢,同时也影响着植物次生代谢产物的积累。苯丙酸盐途径是植物中非常重要的次生代谢途径,4-香豆酸:辅酶A连接酶(4CL)位于该途径中的分支点上,是控制碳流进入不同苯丙酸盐代谢支路的关键酶。4CL蛋白及其基因在木质素合成调控中的研究较多,而在类黄酮的合成调控中的研究较少,甚至其对类黄酮合成的调控机理都不是十分清楚。本文以拟南芥野生型(Col-4)和光受体突变体为材料,系统地研究了蓝光对4CL3的表达和类黄酮合成的调控分子机制,以及初步研究了4CL3在拟南芥中的其它生理功能。论文的具体研究结果如下:(1)通过对拟南芥各种光受体突变体木质素含量的测定及茎的组织化学分析结果发现,当红光和远红光受体光敏素(PhyA,PhyB)突变后,植株的木质素含量降低;当蓝光受体隐花素(CRY1,CRY2)突变后,植株的木质素含量升高。该结果说明,光敏素(PhyA,PhyB)对拟南芥木质素合成起正调控作用,隐花素(CRY1,CRY2)对拟南芥木质素合成起负调控作用。(2)拟南芥幼苗在不同光质处理下,通过半定量RT-PCR和QPCR对苯丙酸盐代谢途径的相关酶基因的表达分析,结果发现4CL3的表达受各种光质的诱导明显,尤其受蓝光的诱导最为强烈,并且与已知的类黄酮合成的关键酶基因CHS表达趋势基本一致。同时还发现,在蓝光受体突变体中,4CL3的表达量显著降低,特别是在蓝光受体双突变体cry1cry2的降低幅度最大。另外,通过4CL::GUS在不同光质下的GUS活性的检测分析发现,4CL3的表达受不同光质的不同程度的正调节,尤其是受蓝光的正调节非常明显。随后,通过对各种光受体突变体在黑暗和蓝光下的类黄酮含量的检测发现,在蓝光条件下野生型中的三种黄酮苷元含量都比在黑暗条件下成倍增加,而且比各光受体突变体中的含量高出几倍,结果说明蓝光受体隐花素(CRY1和CRY2)和光敏素PhyA(吸收部分蓝光)介导蓝光促进了类黄酮的生物合成和在植物体内的积累。这些结果证实,蓝光促进拟南芥4CL3的表达和类黄酮的累积。(3)通过对Col-4、蓝光受体突变体(cry1,cry2,cry1cry2)和过表达转基因株系(35S::GFP-CRY1,35S::GFP-CRY2)中的以对-香豆酸辅酶A为中心呈十字形排列于苯丙酸盐代谢途径中的各种酶基因在持续蓝光下的表达分析,发现主要是4CL3和CHS的表达受由CRY介导的蓝光信号传导途径的诱导非常明显。同时还发现,4CL3的表达和类黄酮的累积主要受CRY1介导的蓝光信号传导途径的正调节,而CRY2起协同作用。(4)通过采用可控条件限制的核定位方法对CRY1进行的研究,当用DEX处理后35S::CRY1-GR中4CL3和CHS的转录水平成倍增加,并达到与35S::GFP-CRY1的同一水平,说明CRY1在核内正调节4CL3和CHS的表达。该结果证明了4CL3和CHS受核内CRY1的正调节,而不受胞质CRY1的调节。同时通过类黄酮含量的检测发现,当有DEX存在时,35S::CRY1-GR中类黄酮含量均成倍增加。该结果说明,CRY1在核内促进了类黄酮的合成。另外,通过检测35S::GFP-CRY1中CRY1与GFP形成的融合蛋白的绿色荧光强度和亚细胞定位发现,蓝光可以促进CRY1由细胞质向细胞核转移。根据这些实验结果我们可以得出这样的初步结论,当植物接受到蓝光时促进CRY1由细胞质进入细胞核,然后在细胞核内促进4CL3和CHS等类黄酮合成的关键酶基因的表达,从而促进类黄酮的合成和累积。根据这些实验结果,我们初步揭示了蓝光对类黄酮合成代谢进行调控的分子机理。(5)在研究中我们发现一个有趣的现象,不论在何种处理条件下,4CL3的表达趋势总是与CHS高度一致,并且4CL3的表达量始终与类黄酮的含量呈正相关性。由此我们推测4CL3不参与木质素的合成,很可能参与类黄酮的合成,是类黄酮合成的关键酶。随后通过对4CL3缺失突变体S894的研究发现,4CL3缺失或表达量的降低会导致植株中类黄酮含量的降低。该结果首次以直接证据证实了4CL3是参与类黄酮合成的关键酶。同时我们也发现,当4CL3缺失仅导致突变体类黄酮含量下降20-40%,并没有出现完全缺失的结果。因此可以推测,4CL3并非是苯丙酸盐代谢途径中催化香豆酸盐形成相应的CoA硫酯用于类黄酮合成的唯一的酶,而只是起主要作用,当其缺失时其它4CL酶(4CL1,4CL2和4CL5)也能行使部分催化功能。因此,我们认为4CL3催化通路是类黄酮合成的主要通路,并且是苯丙酸盐代谢途径中控制碳流进入类黄酮代谢途径的关键酶。根据这些实验结果,我们修正了苯丙酸盐代谢途径。(6)通过对4CL3在长日照(16 h光照/8 h黑暗)或短日照(8 h光照/16 h黑暗)光周期条件下4CL1和4CL3表达变化的生物节律性分析发现,4CL基因家族中已知的参与木质素合成的4CL1具有一定的昼夜节律性,但是不是特别明显。相反,4CL3的表达具有明显的昼夜节律性,即使转移至白光下2 d后仍然保持着这种节律性。该实验结果证明了参与类黄酮合成的4CL3的表达具有明显的昼夜节律性。(7)通过对4CL3的缺失突变体S894和过表达转基因株系35S::GFP-4CL3的表型分析发现,与野生型相比S894的开花时间稍微提前,而35S::GFP-4CL3的开花时间却比Col-4延迟了约6-10 d。另外还发现,S894的植株较野生型Col-4矮小,而35S::GFP-4CL3的植株明显比Col-4高大。这些结果初步证明4CL3对拟南芥开花时间和生物产量有着重要的调节作用。综上所述,本研究从分子水平上初步阐明了隐花素CRY1介导的蓝光信号传导途径对4CL3的表达和类黄酮合成的调控机制,首次以直接证据证实了4CL3是参与类黄酮合成的关键酶,并修正了植物次生代谢途径中重要的苯丙酸盐代谢途径,为将来利用基因工程方法调控类黄酮和木质素的合成提供了理论依据,具有重要的理论和现实指导意义。更多还原

【Abstract】 Light illumination strongly influences not only the primary metabolism of plants, but also the accumulation of secondary metabolites. The phenylpropanoid pathway is a very important secondary metabolic pathway in plants. The 4-coumarate:CoA ligase (4CL) located in the pathway branch point is the key enzyme to control the carbon flow into the various branches of phenylpropanoid pathway. Although extensive research works have been carried out on the regulations of lignin biosynthesis by 4CL protein and gene, much fewer works have been devoted to the flavonoid biosynthesis. As a result, even its regulation mechanism is not very clear. In this paper, the wild-type (Col-4) and photo receptor mutants of Arabidopsis thaliana were used as the experimental materials. The regulation molecular mechanism of the expression of 4CL3 and flavonoid biosynthesis by blue light was systematically studied. Moreover, a preliminary study on other physiological functions of 4CL3 was also carried out. The results were listed as follows:(1) Through the analysis of lignin content and the histochemical assay of stems in the various Arabidopsis photo receptor mutants, it was found that the mutation of the red light and far-red light receptor Phytochrome (PhyA, PhyB) decreased the lignin contents; while the mutation of the blue light receptor cryptochrome (CRY1, CRY2) increased the lignin contents. It suggested that Phytochrome (PhyA, PhyB) played a positive role in the regulation of lignin biosynthesis in Arabidopsis, whereas the cryptochrome (CRY1, CRY2) played a negative role.(2) The Arabidopsis seedlings were treated with different light qualities, and the expressions of the related enzyme genes in the phenylpropanoid pathway were analyzed by semi-quantitative RT-PCR and QPCR. The results showed that the expression of 4CL3 was induced apparently by different light qualities, particularly by blue light. The expression trend of 4CL3 was basically similar to that of CHS, a known key enzyme gene in flavonoid biosynthesis. Moreover, it was found that the expression of 4CL3 reduced significantly in the blue light receptor mutants, especially in the double-mutant cry1cry2 which showed the maximum reduction. The histochemical assay of GUS activity in the 4CL::GUS treated with different light qualities indicated that the expression of 4CL3 was regulated at different levels by different light qualities, with blue light having the most significant positive regulation effects. Subsequently, the flavonoids contents of different photo receptor mutants under dark and blue light conditions were analyzed. The contents of three flavonoid aglycones of wild type in blue light increased several times compared with those kept in dark, and were several times higher than those of photo receptor mutants. These results showed that the blue light receptor cryptochrome (CRY1 and CRY2) and Phytochrome PhyA (part of blue light absorption) mediated blue light to promote flavonoid biosynthesis and accumulation in plants. Therefore, it was confirmed that the blue light promoted 4CL3 expression and flavonoids accumulation in Arabidopsis thaliana.(3) The expressions of the various enzyme genes (taking the p-coumarate:CoA as the center and located in the phenylpropanoid pathway in the form of a cross) in the Col-4, blue light receptor mutants (cry1, cry2, cry1cry2) and over-expression transgenic lines (35S:: GFP-CRY1, 35S:: GFP-CRY2) under continuous blue light were analyzed. The results showed that 4CL3 and CHS were the main genes whose expression were obviously induced by CRY-mediated blue light signal transduction pathway; and 4CL3 expression and flavonoids accumulation were positively regulated mainly by the CRY1-mediated blue light signal transduction pathway and synergistically by CRY2.(4) A conditional nuclear localization approach was employed to investigate the exact subcellular compartment where CRY1 action and regulation take place. When 35S:: CRY1-GR was treated with DEX, the transcription levels of 4CL3 and CHS increased several times and reached the same levels of 35S:: GFP-CRY1, indicating that CRY1 in the nucleus positively regulated the expression of 4CL3 and CHS. It demonstrated that the expression of 4CL3 and CHS were positively regulated by nuclear CRY1 rather than cytoplasmic CRY1. The experimental results also showed that the flavonoids content in 35S::CRY1-GR increased several times in the presence of DEX. All these results suggest that CRY1 in the nucleus promoted the flavonoid biosynthesis. In addition, the results obtained during the analysis of the green fluorescence intensity and subcellular localization of the GFP-CRY1 fusion proteins in 35S:: GFP-CRY1 revealed that blue light can promote CRY1 to transfer from the cytoplasm to the nucleus. Based on these results we can draw the preliminary conclusion that when the plants receive the blue light, CRY1 is promoted to transfer from the cytoplasm into the nucleus, and then the nuclear CRY1 induces the expressions of the key enzyme genes such as 4CL3 and CHS in the flavonoid biosynthesis pathway and thereby promotes flavonoid biosynthesis and accumulation in plants. Based on these experimental results, we preliminarily revealed the regulation molecular mechanism of flavonoid biosynthesis metabolism by blue light. (5) In this study, an interesting phenomenon was found that the expression trend of 4CL3 always had a high degree of consistency with CHS, regardless of the conditions in which the seedlings were treated. There was a positive correlation between the expression of 4CL3 and the flavonoids content. So it was speculated that 4CL3 did not participate in the lignin biosynthesis, it might be involved in the flavonoid biosynthesis and was the key enzyme of flavonoid biosynthesis. The analysis of the 4CL3 mutant S894 showed that the absent or reduced expression of 4CL3 in plants would lead to the reduction of flavonoids content. It was the first direct evidence confirming that 4CL3 is a key enzyme of flavonoid biosynthesis. At the same time, the experimental results revealed that the 4CL3 deletion resulted in only 20-40% reduction of flavonoids content rather than completely absence of flavonoids. Therefore, one can speculate that 4CL3 is not the only enzyme but plays a major role in the conversion of p-coumaric acid into the corresponding CoA ester for the flavonoid biosynthesis in the phenylpropanoid pathway. When the function of 4CL3 is lost, other 4CL enzymes (4CL1, 4CL2 and 4CL5) are also able to execute the part of the catalytic function. Therefore, it can be concluded that the 4CL3 catalytic route is the main channel of flavonoid biosynthesis, and 4CL3 is the key enzyme controlling the carbon flow into the flavonoid biosynthesis branch in the phenylpropanoid pathway. Based on these results, the phenylpropanoid pathway has been modified.(6) The diurnal or circadian rhythmic expression of 4CL1 and 4CL3 genes under both long-day (16-h light/8-h dark) and short-day (8-h light/16-h dark) photoperiods were investigated. It was found that among the 4CL gene family, 4CL1 (a known gene participating in the lignin biosynthesis) had a diurnal or circadian rhythm but not very significant. In contrast, 4CL3 showed the most significant and robust diurnal or circadian rhythm that could even sustain in seedlings transferred from the photoperiod conditions to continuous white light for at least 2 d. These results demonstrated that the 4CL3 involved in the flavonoid biosynthesis had the obvious circadian rhythm.(7) Phenotypic analysis of the 4CL3 mutant S894 and the 4CL3 overexpression transgenic lines 35S::GFP-4CL3 revealed that S894 flowered a little earlier, compared with the wild-type, while the flowering time of 35S:: GFP-4CL3 was delayed about 6-10 d. Moreover, the plant stature of S894 was shorter and smaller than the wild-type Col-4, whereas the plant stature of 35S:: GFP-4CL3 was significantly higher and bigger than Col-4. These results indicated that the Arabidopsis 4CL3 played an important role in the regulation of the floral initiation and biomass production.In conclusion, our studies preliminarily clarified at the molecular level the regulation molecular mechanism of the 4CL3 expression and flavonoid biosynthesis by CRY1-mediated blue light signal transduction pathway, provided the first direct evidence confirming that 4CL3 was a key enzyme of flavonoid biosynthesis, and modified the phenylpropanoid pathway, a very important secondary metabolic pathway in plants. The results obtained during this research project provide a theoretical basis for the controlling of flavonoids and lignin biosythesis by the genetic engineering method, which has an important theoretical and practical significance.更多还原