【作者】 张彬；

【导师】 陈国平；

【作者基本信息】 重庆大学 ， 植物学， 2011， 博士

【摘要】 花青素是一类广泛存在于植物中的水溶性天然色素,属于类黄酮化合物的一种。它存在于植物表皮细胞的液泡中,使高等植物的花、果实、种皮呈现出红、蓝、紫的颜色。在植物中花青素行使着多种重要的生理功能,例如它可以吸引昆虫传粉;防止植物受到紫外辐射,起到UV屏障的作用,保护植物DNA不受破坏、使细胞分化和其它生命过程正常进行;抵御低温以及防止植物病害等。另外大量研究表明花青素具有抗氧化、抗突变、预防心脑血管疾病、保护肝脏、抑制肿瘤细胞发生等多种生理功能。因此,对花青素合成代谢途径以及其调控机制的深入研究,对于提高植物中花青素含量、改良观赏植物的花色、利用生物技术手段开发高含量花青素食品以满足人们日益增长的对健康功能性食品需求都具有重要意义。植物中花青素的合成主要受花青素合成结构基因和调节基因的调控。调节基因编码的转录因子可以调控结构基因的时空表达。在花青素转录调控过程中有WD40、WRKY、BZIP、MADS-box、R2R3-MYB、basic helix-loop-helix (bHLH)等各种转录因子。其中以MYB转录因子、bHLH转录因子和WD40最为重要。此外,植物中花青素的生物合成还受到各种环境因子的调节。影响花青素合成及呈色的外界环境因子主要包括光、温度等。本文以羽衣甘蓝红鸽和白鸽、紫甘蓝和青甘蓝、红菜薹和小白菜为材料,对芸薹属中三种富含花青素的突变体:羽衣甘蓝红鸽、紫甘蓝、红菜薹中花青素合成的分子调控机理,以及不同环境因子对它们花青素合成的影响进行了深入研究,主要研究结果如下:⑴芸薹属植物中花青素合成调控途径关键转录基因的克隆和序列分析:在羽衣甘蓝红鸽和白鸽中分别获得了CRY1、COP1、HY5、PAP1、PAP2、MYB114的全长cDNA序列,它们分别包含了2034bp、2031bp、495bp、750bp、744bp和324bp的开放阅读框序列,并各自编码了680、677、164、249、248、107个氨基酸。蛋白质序列比对分析结果显示,这几个基因编码的蛋白质序列在这两个品种中是完全一致的。在紫甘蓝和青甘蓝中分别获得了CRY1、COP1、HY5、PAP1的全长cDNA序列,它们分别包含了2034bp、2031bp、495bp、753bp的开放阅读框序列,并各自编码了680、677、164、250个氨基酸。蛋白质序列比对分析结果显示,CRY1、COP1基因编码的蛋白质序列在这两个品种中是完全一致的。而HY5和PAP1基因编码的蛋白质序列在这两个品种中各只有一个氨基酸的差异。在红菜薹和小白菜中分别获得了CRY1、COP1、PAP1的全长cDNA序列,以及在红菜薹中获得了PAP2的全长cDNA序列,它们分别包含了2034bp、2031bp、753bp、744bp的开放阅读框序列,并各自编码了680、677、250、248个氨基酸。蛋白质序列比对分析结果显示, COP1和PAP1基因编码的蛋白质序列在这两个品种中是完全一致的。而基因CRY1编码的蛋白质序列在这两个品种中有两个氨基酸的差异。⑵羽衣甘蓝红鸽(Brassica Oleracea var. acephala f. tricolor)是羽衣甘蓝中的一种突变体,其植株在经历低温后,中心部分能够累积大量的花青素而呈现深紫色。为了研究羽衣甘蓝中花青素生物合成代谢调控的分子机理,本试验对比分析了羽衣甘蓝红鸽和白鸽中花青素合成结构基因和相关转录因子的表达情况。结果显示,除F3H在羽衣甘蓝白鸽中的表达量要高于红鸽外,其它的结构基因在红鸽中的表达明显高于白鸽。尤其对于处于下游的结构基因DFR和ANS,它们在羽衣甘蓝白鸽中几乎没有表达。转录因子中,R2R3 MYB转录因子BoPAP1在两个品种间的的表达差异最大,相差达到125倍。且BoPAP1的表达明显是受低温诱导的。序列分析表明BoPAP1是拟南芥中AtPAP1的同源基因。另外,羽衣甘蓝红鸽中花青素的积累是受低温环境胁迫诱导的,结构基因C4H、F3H、DFR、ANS、UFGT在低温条件下的表达水平明显高于室温条件下。以上结果说明BoPAP1在低温条件下促使花青素生物合成途径结构基因过量表达,从而诱导羽衣甘蓝红鸽植株花青素大量积累的过程中发挥着重要的调控作用。⑶紫甘蓝(Brassica oleracea var. capitata)是十字花科、芸薹属甘蓝种中的一个变种。因其外叶和叶球富含有大量的花青素而呈现为紫红色得名。为了研究紫甘蓝中花青素生物合成代谢调控的分子机理,本试验对比分析了光照和黑暗处理条件下紫甘蓝和青甘蓝幼苗中花青素合成结构基因及相关转录因子的表达。结果显示,除C4H和F3H外,紫甘蓝中CHS、ANS、DFR无论在光照还是黑暗条件下的表达水平都明显高于青甘蓝幼苗,其中ANS和DFR的差异尤为明显。说明ANS和DFR是紫甘蓝中花青素合成的两个关键酶基因。进一步对转录因子的研究发现,MYB转录因子BoPAP2与bHLH转录因子BoTT8与结构基因的表达有着相同的表达趋势,即在紫甘蓝中的表达水平要显著高于青甘蓝,且这两个转录因子在黑暗条件下的表达量仍处于较高水平,只是稍低于光照条件。这与紫甘蓝在黑暗条件下仍然能够积累少量的花青素而呈现浅红色的表型现象相吻合。以上结果说明,BoPAP2和BoTT8的过量表达是促进紫甘蓝中花青素合成结构基因表达,导致其花青素积累的主要原因。⑷红菜薹(Brassica campestris L. ssp. chinensis L. var. purpurea Bailey)是白菜种中小白菜亚种的一个变种,是一种高含量花青素天然突变体。为了研究红菜薹中花青素生物合成代谢调控的分子机理,本研究对比分析了红菜薹和小白菜茎表皮与肉质、以及光照和黑暗处理条件下红菜薹和小白菜幼苗中花青素合成结构基因和相关转录因子的表达情况。结果显示,结构基因CHS、F3H、ANS、DFR在红菜薹茎表皮中的表达量要明显高于其肉质部分、以及小白菜的相同部位,这与红菜薹只在茎皮部分积累花青素的表型是一致的。对幼苗的光照和黑暗处理实验中同样也证明了光照诱导以上四个结构基因的过量表达,导致只有光照条件下的红菜薹幼苗才能累积花青素而呈现紫红色。CHS、F3H、ANS、DFR编码的调节酶对红菜薹的花青素合成起到关键的调节作用。进一步对转录因子的研究发现,bHLH家族的一个与花青素合成相关的转录激活因子BrTT8在红菜薹花青素合成的调控中起到重要的调控作用。BrTT8与CHS、F3H、ANS、DFR这四个关键结构基因的表达模式一致,即在红菜薹中花青素积累的组织中都有高丰度的表达,而在红菜薹没有花青素积累的组织以及小白菜中的表达量非常低。⑸在红菜薹和小白菜中分别获得了MYB1基因的全长cDNA序列,它们包含了1164bp的开放阅读框序列,并编码了387个氨基酸。蛋白质序列分析结果显示这两者的氨基酸序列在五处位置上存在差异。为了进一步研究BrMYB1基因的功能,构建了红菜薹BrMYB1p基因的RNAi载体并转化红菜薹,获得了6株转基因植株。观察后发现,转化后的红菜薹外植体在愈伤阶段就变为了绿色,发育为植株后,其茎和叶片都呈现为绿色。通过以上结果可以初步判定BrMYB1p基因参与调控了红菜薹中花青素的合成。更多还原

【Abstract】 Anthocyanins are natural pigments belonging to the flavonoid family. They are responsible for blue, purple, red colors in most flowers, fruits and seeds of higher plants. In plants, anthocyanins are believed to have a variety of functions, including defence and protection against light stress, and the pigmented anthocyanin compounds play a physiological role as attractants in plant/animal interactions. In addition, anthocyanins also have important implications in the field of human health. For example, they can reduce the incidence of cardiovascular disease, cancer, hyperlipidemias and other chronic diseases through the intake of anthocyanin-rich foods. In order to increase anthocyanin content in ornamental plants to increase its aesthetic value or to develop new anthocyanin-rich foods by biotechnology to meet the demand for our dietary consumption of anthocyanins, it is critical to have a complete understanding on the regulation of anthocyanin biosynthesis. The anthocyanin biosynthesis is mainly controlled by structural and regulatory genes. The regulatory genes encoding transcription factors control the temporal and spatial expression of the structural genes. There are numerous transcription factors responsible for regulating the anthocyanin biosynthetic pathway, such as WD40, WRKY, BZIP, MADS-box, R2R3-MYB and basic helix-loop-helix (bHLH). Among these , MYB、bHLH、WD40 have been extensively analyzed and shown to play the most important role in controlling the pathway. Anthocyanins are not always synthesized under normal growth conditions. The biosynthesis of anthocyanin is always affected by environment factors, such as light, low temperature and nutrient depletion.In this study, to investigate the mechanism of anthocyanin biosynthesis in the plant, we compared six cultivars in Brassica , the purple kale“Red Dove”and the white kale“White Dove”, the red cabbage and the green cabbage, the Purple Flowering Stalk and the Pakchoi respectively. The purple kale, the red cabbage, and the Purple Flowering Stalk which are three mutations in Brassica have the strong ability to accumulate anthocyanin pigments. Meanwhile, the mechanism of abiotic stress-induced anthocyanin accumulation were also studied in these cultivars. The main results are as follows:⑴The cloning and analysis of anthocyanin related transcriptional factors in Brassica:The genes of CRY1, COP1, HY5, PAP1, PAP2, MYB114 were separately cloned from the purple kale and the white kale. They contained an open reading frame of 2034bp, 2031bp, 495bp, 750bp, 744bp and 324bp, and encoding 680, 677, 164, 249, 248, 107 amino-acid sequence respectively. Alignment of the protein sequences showed that these genes were entirely consistent with each other in two cultivars.The genes of CRY1, COP1, HY5, PAP1 were separately cloned from the red cabbage and the green cabbage. They contained an open reading frame of 2034bp, 2031bp, 495bp, and 749bp, and encoding 680, 677, 164, 250 amino-acid sequence respectively. Alignment of the protein sequences showed that CRY1 and COP1 were entirely consistent with each other in two cultivars, while there were only one different amino-acid in protein sequences of HY5 and PAP1 respectively beween two cultivars. The genes of CRY1, COP1, PAP1 were separately cloned from the Purple Flowering Stalk and the Pakchoi and the genes of PAP2 was cloned from the Purple Flowering Stalk. They contained an open reading frame of 2034bp, 2031bp, 749bp, and 744bp, and encoding 680, 677, 250, 248 amino-acid sequence respectively. Alignment of the protein sequences showed that COP1 and PAP1 were entirely consistent with each other in two cultivars, while there were only two different amino-acid in protein sequences of CRY1 beween two cultivars.⑵The purple kale (Brassica Oleracea var. acephala f. tricolor) is a mutation in kales, giving the mutant phenotype of brilliant purple color in the interior. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the purple kale, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the purple cultivar“Red Dove”and the white cultivar“White Dove”. The result showed that nearly all the anthocyanin biosythesic genes showed higher expression levels in the purple cultivar than in the white cultivar, especially for DFR and ANS, they were barely detected in the white cultivar. Interestingly,the fact that a R2R3 MYB transcription factor named BoPAP1 was extremely up-regulated in the purple kale and induced by low temperature attracted our attention. The sequence analysis showed that BoPAP1 shared high similarity with AtPAP1. In addition, the anthocyanin accumulation in the purple kale is strongly induced by the low temperature stress. The expression of anthocyanin biosynthetic genes C4H, F3H, DFR, ANS and UFGT were all enhanced under the low temperature. These evidences strongly suggest that BoPAP1 may play an important role in activating the anthocyanin structural genes for the abundant anthocyanin accumulation in the purple kale. ⑶The color of red cabbage(Brassica oleracea var. capitata)is due to anthocyanin accumulation. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the red cabbage, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the seedlings of the red cabbage and the green cabbage treated by light and dark respectively. Except C4H and F3H , the expression levels of the structural genes in the red cabbage were all significantly higher than the green cabbge under not only light treatment but also dark treatment. The increased expression of the structural genes coincided with a coordinated increase in transcript levels of a MYB TFs, BoPAP2 and a bHLH TFs, BoTT8. In addition, the expressions of BoPAP2 and BoTT8 were not down-regulated by dark treatment. This may be the reasons that the red cabbage were still able to accumulate a small amount of anthocyanins under dark treatment. These results indicated that activation of these two regulatory genes up-regulate a set of anthocyanin biosynthetic genes leading to the anthocyanin accumulations in red cabbage.⑷The Purple Flowering Stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey) which is a mutation of Pakchoi contains high levels of anthocyanins, and the accumulations of anthocyanin in it is light-induced. To elucidate the molecular mechanism of the anthocyanin biosynthesis in the Purple Flowering Stalk, we analyzed the expression of structural genes and some transcription factors associated with anthocyanin biosynthesis in the Purple Flowering Stalk and Pakchoi. The structural genes, CHS, F3H, ANS and DFR showed higher expression levels in the stem skin than in the stem flesh of the Purple Flowering Stalk and the stem skin of the Pakchoi. This result was consistent with the phenotype that the anthocyanin was only accumulated in the skin of the Purple Flowering Stalk. Comparing the expression profiling of anthocyanin structural genes in the seedlings of the Purple Flowering Stalk and the Pakchoi treated by light and dark respectively, it showed that CHS, F3H, ANS and DFR were all extremely up-regulated by light leading to the pigmentation in the seedlings of the Purple Flowering Stalk under light treatment. Further studies showed that the increased expression of the structural genes coincided with a coordinated increase in transcript levels of a bHLH TFs, BrTT8.These results indicated that BrTT8 which could be induced by light played an important role in regulating the anthocyanin biosynthesis in the Purple Flowering Stalk.⑸The genes of MYB1 were separately cloned from the Purple Flowering Stalk and the Pakchoi. They contained an open reading frame of 1164bp, and encoding 387 amino-acid sequence. Alignment of the protein sequences showed that there were five different amino-acid in protein sequence of MYB1 beween two cultivars. To further study the fuction of the genes of BrMYB1p, a RNAi expression vector of the target gene BrMYB1p was conducted and transformed into the The Purple Flowering Stalk.There were six transgenetic plants. The color of transgenetic callus and the transgenetic plants were all turn to green. These results revealed that BrMYB1p participated in the regulation of anthocyanin biosynthesis in Purple Flowering Stalk.更多还原