【作者】 田士林；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2014， 博士

【摘要】 辣椒红素是一种天然色素，是红色辣椒果实中类胡萝卜素合成途径的代谢终产物，其含量占成熟期红色辣椒果实色素含量的60%以上。辣椒红素颜色鲜亮，很容易着色，有着较长的保色期，且对人体无毒副作用被大量应用于食品工业，具有很好的发展前景。果实中辣椒红素的形成是由辣椒红素/辣椒玉红素合成酶（Ccs）催化环氧玉米黄质生成的。当果实进入绿熟期后，Ccs基因开始表达并逐渐增强，辣椒红素随之形成并开始积累。研究表明，黄色和橙色的辣椒果实由于缺少Ccs基因或Ccs基因发生了突变，导致辣椒果实成熟时不能变红。目前，辣椒主要在我国的西北部分地区进行大面积种植，这些地区干旱少雨，辣椒生产多采用塑料大棚栽培。而作为大棚的主要成分——塑料棚膜往往多年使用，透光性逐年降低，棚内光照差，一定程度上影响了辣椒色素的生产。随着对辣椒红素的研究不断深入，辣椒红素代谢途径也基本研究清楚，但环境因素对辣椒红素积累的影响目前研究较少，部分环境因素引起辣椒红素含量变化方面的分子机理尚不清楚；如何提高辣椒红素的含量和产量，至今鲜有人报道。基于此，结合前人研究成果，从环境调控入手，针对环境因素引起的辣椒红素合成机理进行分析，阐明影响辣椒红素合成的主要原因及其相关分子机理。主要研究有七部分，结果如下：1.土壤干旱胁迫影响辣椒红素合成及其合成途径关键基因的正常表达。主要包括：不同干旱胁迫下辣椒红素含量的测定以及辣椒红素合成途径相关基因(Ccs、Psy、Lcyb和Crtz)的表达模式分析。结果表明，土壤干旱影响辣椒红素的合成；轻度干旱和正常供水对辣椒红素的影响基本没有差异，但是土壤中度干旱和重度干旱往往会引起辣椒红素含量的降低。其次，干旱胁迫抑制了辣椒红素生物合成途径中关键基因的正常表达，尤其是干旱抑制了Crtz和Lcyb基因的正常表达，也是造成辣椒红素含量降低的原因之一。2.果实套袋影响辣椒红素的积累。主要包括：对不同时期的辣椒果实套袋，果实套袋对果面温度的影响，果实套袋对辣椒红素合成途径关键酶基因的影响。结果表明，套袋影响辣椒红素的合成，特别是在辣椒果实绿熟期套袋对辣椒红素的积累影响尤为突出。另外，套袋影响辣椒红素合成途径中Crtz和Ccs基因的正常表达，是导致套袋降低辣椒红素合成的主要原因之一。3.运用cDNA-AFLP差异表达的方法对遮光引起辣椒果色变化的差异基因进行了筛选。一共获得了80条差异条带(TDFs)。将这些TDFs进行测序和分析，根据遮光引起果皮褪绿的现象，序列比对发现一个与叶绿素相关的差异条带，将其进行电子克隆，测序后获得基因全长。通过蛋白三维结构的预测，发现该基因与辣椒材料Cv.VoucherBlackcluster上的核酮糖1,5-二磷酸盐碳酸酵素(rbcL)基因(GenBank: KF917582.1)同源性较高，是一种单体蛋白。聚类分析也表明该基因和rbcL基因为同一类基因。生物信息学分析发现，该基因可能参与叶绿素的合成过程。利用VIGS技术对该基因进行功能初步鉴定，鉴定结果表明，当沉默该基因时，辣椒果实发生了提前褪绿的现象，而且沉默后该基因的表达水平也大幅度降低；最后再次将辣椒果实进行遮光处理，检测该基因在遮光情况下的表达情况。检测结果发现，该基因在遮光情况下表达量很低，而且遮光后果实中的叶绿素含量也明显减少，进一步证明该基因的确参与了辣椒果实中叶绿素的合成过程，属于光敏感型基因。这一结果的获得，阐明了在弱光环境条件下辣椒红素含量降低的同时辣椒果色所出现的提前褪绿并转红的分子机理。4.适当浓度的ABA处理能够促进辣椒红素合成。主要包括，研究影响辣椒红素合成的外源ABA水溶液的最佳浓度；外源ABA的施用对辣椒红素合成途径关键酶基因的调控；外源ABA的喷施对内源ABA合成途径相关基因表达情况的影响等。结果表明，与对照相比，150mg/L浓度下的外源ABA对辣椒红素的合成有促进作用；同时外源ABA的施用也使ABA代谢途径中的关键基因(AO、Zep、NCED)高水平表达。外源ABA的施用，刺激了辣椒红素合成途径中Ccs基因及ABA合成的限速酶基因NCED的长时间持续高效表达，直接导致辣椒红素和ABA含量的增加。而随着内源ABA含量提高增加了辣椒果实的抗性，从而间接地保障了辣椒果实的健康地生长。5.进一步研究了Ccs基因与辣椒果实红色的关系。具体包括：不同果色辣椒Ccs基因的检测；在果实转色期，Ccs基因表达情况分析；以及不同果色Ccs基因开放阅读框的基因序列的比较。结果发现，Ccs基因并不仅在红色辣椒果实中表达，它也存在于部分黄色和部分橙色辣椒果实中。通过对红色辣椒R15、橙色辣椒R37-1和黄色辣椒CK7中的辣椒红素合成途径各基因的表达情况进行分析发现，虽然辣椒红素的合成受Ccs基因调控，但同时也受其他基因的调控，多个相关基因的协同配合，才能保证辣椒红素的顺利合成。6.建立了离体辣椒果实果色调控基因的VIGS检测体系。在前人研究的基础上，以离体辣椒果实为试材，通过烟草脆裂病毒诱导辣椒果实颜色形成相关基因沉默，构建重组病毒沉默载体pTRV2-Psy, pTRV2-Lcyb, pTRV2-Crtz, pTRV2-Ccs；利用农杆菌注射法，对辣椒红素合成途径的关键基因逐个或同时沉默，观察辣椒果实颜色的变化，解释不同基因在辣椒果实红色形成中的作用，为辣椒果实的果色调控提供了技术支持。7.探讨了调控辣椒红素合成的Ccs基因上游启动子不同重复单元的功能。通过构建1个启动子基本单元和3个不同重复单元启动子植物表达载体，采用农杆菌介导的瞬时表达方法，对这些不同重复单元启动子活性进行分析。GUS染色分析发现：与阴性对照相比，PL0，PL1，PL2，PL2-1启动子序列均有活性，而且活性差异不大；而基本启动子单元PL3活性较弱。初步判断Ccs基因启动子中的顺式作用元件不是剂量效应而是位置效应，即一个Ccs基因启动子的重复单元就能起到对Ccs基因的转录和调节转录的效果。更多还原

【Abstract】 Capsanthin is a natural edible red pigment, it is a metabolic end product in red pepperfruits carotenoid biosynthetic pathway, which account for60%of the content of mature fruitpigment. Capsanthin has the virtue of brilliant color and good staining function. It canenhance antistaling time of food, non-poisonous effects on God’s image. Thus, it will replacechemical synthesis of pigment by degrees in the near future, and will be applied widely in thefood processing department. Capsanthin/capsorubin synthase (Ccs) catalyzes zeaxanthin toform capsanthin. The expression of Ccs gene is undetectable in immature pepper fruit. Whenpepper fruits begain to being green mature, and Ccs gene began to express and graduallyincreased, capsanthin subsequently formed and continuously accumulate. Research indicatedthat capsanthin can not be synthesized because of mutation or deletion of Ccs gene in yellowor orange pepper fruits, so, the ripened fruits could not turn red.Currently, peppers (Capsicum) are widely cultivated as a natural pigment resource inparts of Chinese north-west regions. The lack of rain aggravated pepper production in theseareas. So, plastic greenhouse cultivation becomes a major planting mode of pepper production.But plastic films as the main component of greenhouses are often used for many years, andthe rate of light permeation of plastic films is declining by degrees, with poor light conditionsin plastic sheds. All of these will affect pepper pigment production.With the development of capsanthin research, the biosynthesis pathway of capsanthin isclear basically, but it was few concern how environmental factors affect the synthesis ofcapsanthin. The molecular mechanism of environment effect on capsanthin synthesis is notclear. From that point, and jointing predecessor research results, we researched capsanthin bylearning environmental regulation to find out the molecular mechanism of capsanthinsynthesis, and to explain how environment factors affect the synthesis of capsanthin. Themain research conclusions include seven parts, as below:1. Soil drought stress affected capsanthin accumulation and expression of capsanthinbiosynthesis-re-related genes. Mainly including: capsanthin was determined under differentdegree stress; and the expression of capsanthin synthesis pathway genes (Ccs, Psy, Lcyb andCrtz) was analyzed. The results showed that drought significantly affected capsanthin synthesis in pepper fruits; there was no significant difference between the control and LDgroups, however, moderate to severe drought had the greatest impact on capsanthinaccumulation. Analyzing the expression of key genes in the capsanthin biosynthesis pathwayindicated that soil drought inhibited key genes express; especially, soil drought has a greatereffect on the expression of the lycopene-β-cyclase (Lcyb) and β-carotene hydroxylase (Crtz)genes, resulting in a significant reduction in the fruit capsanthin content in pepper.2. Fruit bagging affected capsanthin synthesis in the pepper. Mainly including: effect offruit bagging on fruit surface temperature and capsanthin synthesis in the stage of pepper fruitdevelopment; effect of pepper fruits bagging on the expression of capsanthinbiosynthesis-re-related genes in the stage of pepper fruit development. These researchconclusions showed that bagging inhibited capsanthin synthesis in the stage of pepper fruitdevelopment; of note, bagging had the greatest effect on capsanthin during the mature-greenstage of the fruit. Bagging restrained the accumulation of capsanthin, and the chief reason wasthat bagging inhibited Crtz and Ccs genes expression over the entire course of pepper fruitdevelopment and ripening, which may be the main reason that fruits bagging affectedcapsanthin synthesis.3. Using cDNA-AFLP technique, some differently expressed genes that fruit shadingcaused fruit color change were screened. We obtained and identified80transcript derivedfragments (TDFs), these TDFs were sequenced and analyzed. According to the phenomenonthat shading caused peel faded green, a TDF associated with chlorophyll was found bysequencing alignment. The full-length gene was obtained by electronic cloning andsequencing. By means of homologous protein three-dimensional structure prediction method，it was found that the gene was quite similar to the ribulose1,5-diphosphate enzyme carbonicacid (rbcL) gene of Cv.Voucher Blackcluster (GenBank: KF917582.1), and the protein of thisgene is a monomeric protein. The cluster analysis also showed that the gene and rbcL geneare in the same group. Bioinformatics analysis indicated that the gene may be concerned withthe synthesis of chlorophyll. And named CarbcL, and registered in GenBank (KM489073).Using VIGS technology to analyse CarbcL gene function, identification results showed thatpepper fruits faded green early and turn red when the gene was silenced, and CarbcL geneexpression level decreased strikingly. Finally, we researched CarbcL gene expression level byusing light-interrupted fruits. The results indicated that CarbcL gene expression level wasvery low in the shading; the color of light-interrupted fruits changed from dark green to thingreen; and the content of chlorophyll reduced considerably. These data further confirm thatCarbcL gene was indeed concerned with the synthesis of chlorophyll in pepper fruits, topertain to light-sensitive gene. These experimental results were obtained to clarify the phenomena that pepper fruit color faded green early and turn red under the condition of weaklight, to explain the regulatory mechanism of weak light influencing fruit color in the stage ofpepper fruit development.4. The appropriate concentration of ABA could improve the content of capsanthin inpepper fruits. Mainly including: optimizing optimal exogenous ABA concentration impacttingon capsanthin content; applying exogenous ABA effect on key genes of capsanthinbiosynthetic pathway. The expression level of key genes in ABA biosynthetic pathway whenexogenous ABA was sprayed on pepper fruits. The main conclusions were: the fruitcapsanthin content was increased highly than that of the control by using ABA150mg/Lconcentration. And the expression of capsanthin biosynthesis-re-related genes (Psy, Lcyb,Crtz and Ccs) is increased. And key genes (AO, Zep and NCED) which regulate ABAmetabolic pathway had an elevated expression level too. That lead to capsanthin content wasincreased. The resistant of pepper fruits to abiotic stresses had been improved with theincreasing of endogenous ABA content, it indirectly guaranteed the normal growth of pepperfruits.5. Further explanation was done about the relationship of genes and red fruit colors inpepper. Including concretely: Ccs gene was detected in Capsicum with different fruit colors;Ccs gene expression was analyzed in fruit color-changed Period; comparing the sequences ofCcs gene open reading frame in Capsicum with different fruit color. Our results showed Ccsgene not only exist and express normally in red pepper fruit (cv. R15), but also present andexpress normally in parts of yellow(cv.CK7) and orange (cv. R37-1) pepper fruit. Throughanalyzing the related genes expression of red (cv.R15), orange (cv.R37-1) and yellow (cv. CK7)Capsicum cultivars in capsanthin synthesis pathway，it was found that the synthesis ofcapsanthin was regulated not only by Ccs gene, but also by other genes. It suggested thatcapsanthin biosynthesis is very complex pathway in pepper fruits colors formation and theabnormal expression of any key genes will affect red color formation and capsanthinsynthesis.6. Establishing the VIGS detection system for fruit color-re-related genes in detachedpepper fruits. On the basis of previous studies, in detached pepper fruit was selected as theresearch matierials in this study. We constructed viral vectors (pTRV2-Psy, pTRV2-Lcyb,pTRV2-Crtz, pTRV2-Ccs), using the tobacco rattle virus (TRV) carrying target gene (Psy,Lcyb, Crtz and Ccs genes) to infect detached pepper fruits. By using VIGS technology, onegene of carotenoids synthesis in pepper fruit was silenced or several genes were silencedsimultaneously, and fruit color variation was observed. Explain the role of different genes in the formation of fruit red color. This provided us a technical support to better realize themolecular mechanism of pepper fruit color formation.7. Functions of specific repeat sequences of Ccs gene promoter were discussed. Byligating L0, L1, L2, L2-1and L3DNA fragments and plant expression victor PBI121containing CaMV35s promoter individually to form recombinant vectors PL0, PL1, PL2,PL2-1and PL3. These recombinant vectors (PL0, PL1, PL2, PL2-1and PL3) were transfectedto agrobacterium tumefaciens GV3101. The repeat fragments were analysed by analyzing theactivity of GUS gene. Compared with the control, PL0, PL1, PL2, PL2-1repeat unit had someactivities, and there were no significant differences among PL0, PL1, PL2, PL2-1promoterrepeat units; while PL3had a little activity. It should be preliminary concluded thatCis-element in the promoter of Ccs gene was not a dosage effect but a position effect. That isto say, a repeating unit of Ccs gene promoter can play a transcription and regulation role inCcs gene expression.更多还原