【作者】 李艳军；

【导师】 孙杰； 夏桂先；

【作者基本信息】 石河子大学 ， 作物遗传育种， 2013， 博士

【摘要】 1.研究目的彩色棉是一种成熟吐絮时纤维自身带有天然色彩的棉花。近年来，人们的健康和环保意识逐渐增强，天然彩色棉制品倍受人们青睐。然而，彩色棉纤维的颜色仅有绿色和棕色两大色系，颜色单调，远不能满足人们对于绚丽服饰的需求。由于缺乏除棕、绿两种颜色之外的其他颜色的种质资源，通过常规的杂交手段难以解决目前彩色棉颜色单调的问题。人们寄希望利用基因工程手段来改变彩色棉纤维的颜色，完成这项工作的前提是明确彩色棉纤维色素形成的分子机制。本研究利用2-DE结合质谱鉴定分析比较了彩色棉及其白色近等基因系棉纤维蛋白质组的差异，分离鉴定差异蛋白，为揭示彩色棉色素合成的分子机制及为利用基因工程手段改良彩色棉纤维色泽品质提供理论基础。2.结果与结论2.1利用差异蛋白质组学策略分析比较了棕色棉及其白色近等基因系棉纤维蛋白质组差异，利用MALDI-TOF/TOF质谱技术成功鉴定了3个纤维发育时期（12，18和24DPA）共计78个差异蛋白点。研究发现21个差异蛋白点与植物次生代谢途径相关，其中15个参与类黄酮物质的合成，且均在棕色纤维中表现上调；DMACA染色实验以及7个原花色素合成关键酶基因的qPCR分析表明，类黄酮在棕色棉纤维色素形成过程中起关键作用，推测原花色素与纤维颜色形成有关。本研究首次从蛋白质组学的角度明确了类黄酮在棕色棉纤维色素合成过程中的重要作用，也为类黄酮是棕色纤维中的色素物质这一假设提供了直接的证据。同时发现了一些在棕色纤维中下调的蛋白点，包括糖酵解、氧化还原平衡，细胞骨架和蛋白质代谢相关蛋白，推测棕色纤维中类黄酮化合物的活跃代谢影响了其它代谢途径的正常运作，一些纤维发育相关的蛋白，如ascorbate peroxidase、superoxide dismutase、actin、annexin和heat shock protein70等表现下调，是棕色棉纤维品质差的主要原因。2.2利用差异蛋白质组学策略分析比较了绿色棉及其白色近等基因系棉纤维蛋白质组差异，在3个纤维发育时期（12，15和24DPA）成功鉴定了25个差异蛋白点。这些差异蛋白点包括次生代谢、能量/碳水化合物代谢、细胞壁合成、蛋白质代谢、细胞骨架、氨基酸代谢、细胞响应和脂代谢等相关蛋白，其中大部分蛋白点表现为下调。几个差异明显的蛋白点分别为phospholipase D、phenylcoumaran benzylic ether reductase和s-adenosylmethionine synthase，暗示它们在绿色棉色素合成过程中起关键作用。为了进一步寻找绿色纤维中色素合成相关基因，本研究利用双向电泳技术研究了21DPA绿色棉纤维蛋白质组，从凝胶上挖取220个蛋白点进行MALDI TOF/TOF分析，成功鉴定出156个蛋白。根据蛋白的功能将这些蛋白分成12类，主要包括能量/碳水化合物代谢、细胞壁合成、氧化还原平衡和细胞骨架相关蛋白，反映了21DPA绿色棉纤维中的生理活动状态。根据差异蛋白质组策略和21DPA蛋白质组数据，在绿色纤维中没有发现明显的色素合成途径，类黄酮相关蛋白点在绿色纤维中无上调现象，表明绿色纤维中的色素物质与棕色纤维不同。2.3花色素还原酶是合成表儿茶素的关键酶之一，表儿茶素是原花色素的合成单元。利用比较蛋白质组学策略发现了一个在棕色棉纤维中明显上调的蛋白点，质谱鉴定为花色素还原酶。根据GenBank中该蛋白的cDNA序列克隆了GhANR基因，该基因包含1008bp核酸序列，编码336个氨基酸残基。实时荧光定量PCR分析显示GhANR在棕色纤维中的表达量高于白色棉。将GhANR连接至蛋白表达载体pET28a，转化大肠杆菌表达蛋白，酶活性测定表明GhANR蛋白能够催化矢车菊素转化为表儿茶素。更多还原

【Abstract】 1. Object: Naturally colored cotton is cotton endowed with natural colors. Because the color exist in the fiber,dyeing is not required during the fabric manufacturing process. The use of naturally colored cotton couldeliminate dyeing costs and the disposal of toxic dye waste. As concerns for human health and the environmentincrease, naturally colored cotton has become an environmentally friendly option. However, the colors ofnaturally colored cotton primarily include green and several shades of brown. Cross-breeding can only generatenew shades of brown and green, which are insufficient for clothing versatility. Therefore, the color types havenot been altered by conventional breeding due to the lack of genetic resources for the generation of color.Genetic engineering is recognized as a way to generate new fiber color types and has just begun. For the geneticengineering approach, it is important to characterize the genes related to pigment biosynthesis. In this study, weused a proteomic approach to compare the protein profiles of brown color fiber (BCF) and white color fiber(WCF) to identify functional proteins involved in pigment synthesis. The comparison of the protein profiles ofBCF and WCF is of critical significance for the elucidation of the molecular mechanism of pigment biosynthesisand the development of new fiber color types via genetic manipulation.2. Results and Conclusions2.1A comparative proteomic analysis was performed to identify the differences between brown cotton fiber anda white near-isogenic line, and78differential spots were identified at three time points (12-,18-, and24-daypost-anthesis [DPA]) using MALDI-TOF/TOF. Our data illustrate several aspects of pigment synthesis and fiberdevelopment in brown color fiber (BCF). First,21spots were associated with secondary metabolism;15of thesewith high abundance in BCF were involved in flavonoid biosynthesis. DMACA staining and qPCR of7genesencoding enzymes involved in proanthocyanidins demonstrated that a multitude of proanthocyanidins weredeposited in BCF, which, when taken together with our comparative proteomic analysis and previous studies,suggested proanthocyanidins should be the pigment products in BCF and confirmed the crucial role offlavonoids in pigment synthesis in brown color fibers. This is the first time a proteomic approach has providedevidence to support the hypothesis that flavonoids are responsible for pigment synthesis in BCF. Second, severalspots with lower abundance in BCF were found, including glycolytic pathway, redox homeostasis, cytoskeleton,and protein metabolism related proteins. Our results implied that flavonoid synthesis was prevalent, and manymetabolic pathways that are active during fiber development may have been repressed in BCF, which may partlyaccount for the inferior fiber qualities of BCF. The identification of these protein spots with lower abundance inBCF, including proteins that are critical for fiber development, such as ascorbate peroxidase, superoxidedismutase, actin, annexin and heat shock protein, shows molecular evidence for explaining the inferior fiberqualities of color genotypes and provides insights for the fiber quality improvement of color types by geneticmanipulation.2.2A comparative proteomic analysis was performed to identify the differences between green cotton fiber and awhite near-isogenic line, and25differential spots were identified at three time points (12-,18-, and24DPA)using MALDI-TOF/TOF. These proteins were related to secondary metabolism, energy/carbohydrate metabolism,cell wall-related, protein metabolism, cytoskeleton, amino acid metabolism, cell response, lipid metabolism.Several spots with significantly changed abundance were identified as phospholipase D, phenylcoumaranbenzylic ether reductase and s-adenosylmethionine synthase, suggested that they may play very important role inpigment biosynthesis. In addition, the proteome of21DPA (Days Post Anthesis) green cotton fiber was analyzedusing two-dimensional gel electrophoresis to yield a protein map. A total of220individual spots were excisedand analyzed by MALDI-TOF/TOF MS. A total of156proteins were identified and cataloged according to theirfunctions. Twelve different classes of proteins were identified in21DPA green cotton fiber. Many of theseproteins were related to carbohydrate metabolism and energy production. Other notable functional classesincluded oxidoreductases, cell wall-related and cytoskeleton. This study provides insight into the majorphysiological events in21DPA green cotton fiber, and advances our understanding of molecular mechanismsrelated to pigment biosynthesis in green cotton fiber. Although pigment metabolism is an additional process innaturally colored cotton fiber that differs from white cotton fiber, we did not find specific proteins related topigment biosynthesis. In addition, flavonoid biosynthesis related protein spots showed no any changes in abundance, suggesting that the pigment in green cotton fiber was different from that in brown cotton fiber.2.3Anthocyanidin reductase (ANR) is a key enzyme which catalyzes the anthocyanidins into the correspondingepicatechin, an initiating monomer of proanthocyanidin synthesis. Using2D-PAGE comparisons, a protein spotwere found to be significantly up-regulated in brown color cotton fiber but hardly detectable in the white colorcotton fiber. The spots were excised from the gel, partially sequenced and identified to be ANR. GhANR cDNAcontained an open reading frame of1008bp encoding a protein of336amino acid residues. Real-time PCRanalysis showed that the expression level of GhANR was higher in BCF compared with WCF. When GhANR wasexpressed in Escherichia coli, the GhANR protein was showed possessing ANR activity and can catalyze theconversion of cyanidin to epicatechin.更多还原