【作者】 王宇；

【导师】 李玉花；

【作者基本信息】 东北林业大学 ， 发育生物学， 2013， 博士

【摘要】 短波长光质例如蓝光与紫外线是调节植物生长发育重要的环境因子之一。蓝光会影响植物的生长形态如：向光性、叶绿体移动、气孔张开和花青素积累。高强度的紫外线会破坏植物体内的DNA, RNA和蛋白质,而低强度的紫外线会影响植物的生长形态变化、类黄酮类的合成和防御相关基因的表达。然而紫外线调控花青素的信号传递途径至今并未被阐明。本论文以花青素合成光敏感型的津田芜菁(Brassica rapa’Tsuda’)为试材,进行了短波长光质诱导花青素合成特性的研究；CHS家族基因的克隆及短波长光质诱导下的表达特性分析；花青素合成相关R2R3类MYB因子的克隆及其他调节基因在短波长光质诱导下的表达特性分析；花青素合成关键调节因子BrPAP1和BrTT8与BrCHS1启动子区Unit1元件的相互作用分析；不同短波长光质诱导下转录组学分析。得到以下主要结果：1津田芜菁在不同短波长光质诱导下花青素积累特性分析对蓝光、UV-A和UV-B及它们之间的复合光对津田芜菁幼苗下胚轴不同部位花青素的积累进行了分析。不同短波长光质诱导下花青素积累部位并不相同：(1)蓝光诱导花青素合成主要集中在下胚轴的上部；(2)UV-B诱导在上部和中部；(3)UV-A诱导在中下部。同时,蓝光+UV-B复合光的照射会产生增益效应,而其他光质的组合并没有这种现象。UV-A及蓝光+UV-B复合光可以诱导成熟津田芜菁膨大的肉质根表皮花青素积累。2CHS家族基因的克隆及短波长光质诱导下的表达特性分析利用Southern杂交确认津田芜菁基因组中至少存在六个CHS基因拷贝,利用RT-PCR扩增获得BrCHS5和BrCHS6基因的全长克隆。加上本实验室前期工作中克隆获得的BrCHS1-4基因,在获得的六个CHS基因中,BrCHS1,4,5受光诱导表达,而其余三个没有光反应。BrCHSl,4,5特异地受UV-A及蓝光+UV-B复合光诱导在津田芜菁幼苗下胚轴的中下部表达,这与色素积累的部位保持一致。其中BrCHS5的表达量随光照时间延长及光照强度增加而线性积累,而BrCHS4受蓝光+UV-B诱导表达量最高。相反的,BrCHS基因在蓝光诱导下仅在下胚轴上部微弱表达。同时CHS与DFR基因在成熟津田芜菁中的光诱导表达特性与花青素积累特性类似。3花青素合成相关R2R3类MYB因子的克隆及其他调节基因在短波长光质诱导下的表达特性分析利用PCR克隆获得6个可能参与花青素合成R2R3类MYB基因,实时荧光定量PCR结果表明：无论在津田芜菁幼苗还是成熟的肉质根表皮PAP1基因的表达均受光调控,且表达特性与花青素积累特性类似。同时MYB4, MYB12和MYB111基因在不同短波长光质诱导下在幼苗下胚轴不同部位表达模式具有特异性。BrTT8也受光诱导,表达量相对较低,表明其可能作为一个辅助因子参与花青素的合成。4花青素合成关键调节因子BrPAP1和BrTT8与BrCHS1启动子区Unit1元件的相互作用分析通过PCR克隆获得BrCHS4和BrCHS5基因启动子序列,加上本实验室前期工作中克隆获得的BrCHS1基因启动子,利用生物信息学的方法分析得到BrCHSl,4,5启动子区由ACE, RRE和MRE光反应元件组成的顺式作用元件CHS-Unit1。通过酵母单杂交实验证明,津田芜菁BrPAP1、BrTT8和BrHY5因子可以与BrCHS1启动子区CHS-Unit1元件发生特异性的相互作用。5不同短波长光质诱导下的转录组学分析利用RNA-seq技术分析了不同短波长光质处理6小时的津田芜菁转录组的变化。共获得50703个平均长度为1286.44bp的Unigene,其中有847个受UV-A特异诱导的基因,包含花青素合成相关基因CHS、F3H和DFR等,并含有MYB、bHLH和zinc finger等转录因子和其他信号转导因子,分别参与胁迫反应、含硫氨基酸合成代谢反应、应答化学物刺激和色素的合成等过程。利用MEME软件对UV-A特异诱导基因群的启动子区进行保守元件预测分析,获得了一个可能与UV-A反应相关的顺式作用元件,但其功能还需要相关试验进一步的验证。以上结果表明,津田芜菁花青素合成存在三种不同的光诱导反应：UV-A反应、蓝光+UV-B增益效应和蓝光/UV-A反应。UV-A诱导的花青素合成是不同于隐花色素或UV-B受体介导的花青素合成的新途径。更多还原

【Abstract】 Short-wave length light, such as blue and ultraviolet light, plays an important role in plants. Blue light affects are known to affect the morphology of plants, including phototropism, chloroplast migration, stomatal opening and anthocyanin accumulation. Short-wave length (high-energy) UV light can physically damage DNA, RNA and proteins, while UV at low fluence rates were found to induce morphological changes, flavonoid production and the expression of defense-related genes. However, details of UV signaling pathways control anthocyanin synthesis are still unknown.The Brassica rapa’Tsuda’which anthocyanin biosynthesis is light-dependent was used as materials in this research. The investigations were carried out for various patterns of anthocyanin accumulation by different short-wave length light qualities; the cloning and expression of CHS family genes in response to different short-wave length light qualities; the cloning of anthocyanin biosynthesis related R2R3MYB factors and expression pattern of other regulatory genes in response to different short-wave length light qualities; the interactions of BrPAP1and BrTT8with Unitl element of BrCHS1promoter and the analysis of transcriptome in response to different short-wave length light qualities. The main results were obtained as below.1Different anthocyanin accumulation pattern of Brassica rapa’Tsuda’in response to different short-wave length light qualities.The effects of irradiating blue, UV-A, UV-B and a combination of the lights on anthocyanin accumulation at different hypocotyl positions were investigated in the seedlings of turnip ’Tsuda’. The location of anthocyanin accumulation varied depending on different light spectra. Stronger accumulation of anthocyanin was induced (1) at the upper hypocotyl positions by blue light;(2) mainly at the upper position, but also at the middle position by UV-B light; and (3) at the middle to lower position by UV-A light. There were synergistic effects between blue and UV-B, while such effects were not observed for the other light combinations. UV-A and blue+UV-B can induce anthocyanin biosynthesis in the swollen hypocotyls of turnip’Tsuda’.2Cloning and expression of CHS family genes in response to different short-wave length light qualities.Southern blot result showed that there were six copies of BrCHS genes in the genome of turnip’Tsuda’. The full length of BrCHS5and BrCHS6were amplified by RT-PCR. Among the six chalcone synthase (CHS) genes identified in this turnip, BrCHS1,4and5exhibited light-dependent expression patterns, while the other three showed no apparent light responses. The expression of BrCHS1,4and5were increased particularly by UV-A and blue+UV-B irradiation at the middle to lower hypocotyl positions, in accordance with anthocyanin accumulation patterns. BrCHS5increased in a linear manner as irradiation time and light intensity increased. The highest induction of gene expression was observed for BrCHS4upon blue+UV-B co-irradiation. In contrast, CHS expression was induced only slightly at higher hypocotyl positions by blue light. Meanwhile, the expression patterns of CHS and DFR gene induced by light were the same as anthocyanin accumulation in the swollen hypocotyls of turnip’Tsuda’.3Cloning of anthocyanin-related R2R3-type MYB transcription factor genes and expression analysis of regulatory genes in anthocyanin pathway in response to different short-wave length light qualities.Six R2R3-type MYB transcription factor genes were isolated by PCR, and the result of Real-time PCR showed that:PAP1was up regulated by light both in hypcotyls of seedling and swollen hypocotyls of turnip’Tsuda’, and the expression pattern was the same as anthocyanin accumulation. Meanwhile, the MYB4, MYB12and MYB111genes exhibited differential expression patterns at different hypocotyl positions; these patterns were unique for different light spectra. BrTT8genes also induced by light, but had a lower expression. Which indicated that it might be a co-factor participating in anthocyanin biosynthesis.4Two key factors controlling anthocyanin synthesis:interaction of BrPAP1and BrTT8with Unit1element of BrCHSl promoter.BrCHS4and BrCHS5promoters were isolated by PCR, and the CHS-Unit1motif which consisted of the light-responsive element ACE、RRE and MRE in the BrCHS1,4,5promoters was predicted by bio informatics method. Yeast one-hybrid assay showed that BrPAP1, BrTT8and BrHY5had high binding activities to the CHS-Unit1motif of the BrCHSl promoter.5Transcriptome induced by different short-wave length light qualities.Transcriptome changes of6hours’different short-wave length light qualities treatment with Brassica rapa’Tsuda’were analyzed by the RNA-seq.50703Unigene with average length of1286.44bp were obtained. Among them,847were specifically induced by UV-A, included anthocyanin synthesis genes CHS, F3H and DFR, and also contained MYB, bHLH, zinc finger and other signal transduction factors. These genes were involved in response to stress, sulfur amino acid biosynthetic process, response to chemical stimulus and pigment biosynthetic process respectively. One conservative element was predicted from promoter of genes in UV-A-specific cluster by MEME program, but further analysis is needed to confirm the functions of it.These results demonstrate that there is a distinct UV-A response, blue+UV-B synergistic response and blue/UV-A light response for anthocyanin biosynthesis in turnip. UV-A light- dependent anthocyanin biosynthesis appeared to be regulated in a manner that is distinct from that mediated by cryptochromes and UV-B photoreceptors.更多还原