【作者】 庞朝友；

【导师】 喻树迅；

【作者基本信息】 中国农业科学院 ， 作物遗传育种学， 2009， 博士

【摘要】 棉花纤维是从胚珠外表皮细胞分化而来的单细胞结构,是纺织工业的重要原料,具有重要的经济价值。同时,棉纤维细胞也是研究细胞伸长、分化和细胞壁合成等重要生物学现象的理想系统。植物初生细胞壁主要由果胶、半纤维素和纤维素等多糖组成。本研究利用蛋白质双向电泳技术,建立了棉花纤维快速伸长期的银染电泳图谱,发现了101个在棉纤维中高表达的蛋白点。又通过MALDI-TOF MS技术鉴定了其中的93个蛋白点,它们由66个基因编码而成。其中43条cDNA全长是首次报道的。继而,又引入了一种代谢途径分析软件KOBAS用于分析这些纤维优势表达蛋白,发现了17条野生型显著上调的生物代谢途径,其中核苷糖转换代谢途径是纤维伸长过程中最显著上调的生物代谢途径。核苷糖是合成细胞壁多糖的活化底物。利用Nano-LC-FTICR-MS技术进一步鉴定了与核苷糖转换相关的7个蛋白点,确定它们是由四个基因UER1、UGD1、UGP1和UGP2编码而成的。乙烯和C24:0处理的蛋白质组学和转绿水平分析表明:乙烯和24碳饱和脂肪酸(C24:0)能诱导UER1、UGD1和UGP1的蛋白和转录水平的显著提高。暗示二者能够通过诱导某些特殊细胞壁多糖的合成,从而导致纤维细胞的伸长。气相色谱分析细胞壁非纤维素多糖成分发现,与胚珠组织相比,快速伸长的纤维初生细胞壁含有显著多的阿拉伯糖、鼠李糖和半乳糖醛酸,而木糖和葡萄糖较少。在胚珠体外培养的培养液中添加UDP-鼠李糖能显著速进纤维的伸长,而加入鼠李糖对纤维伸长没有影响。UER基因的拟南芥突变体uer1-1根毛变短,并且这种表型能够被拟南芥UER1和棉花UER1所互补。MS培养基中添加UDP-鼠李糖能够回复突变体uer1-1的表型,而鼠李糖则不能。在拟南芥乙烯信号突变体ein2-5和超长链脂肪酸合成突变体cut1植株内,AtUER1基因的表达丰度显著降低。UDP-鼠李糖处理能够回复ein2-5和cut1根毛变短的表型。结果表明乙烯和C24:0能够调控UER的表达,从而导致棉纤维和拟南芥根毛的伸长。蛋白质组学、代谢组学和遗传学证据均表明UDP-鼠李糖的合成对棉花纤维和拟南芥根毛的伸长有重要作用。进一步的分析表明,合成果胶多糖前体核苷糖UDP-阿拉伯糖和UDP-半乳糖醛酸的关键基因GAE1和UXE1在纤维快速伸长期显著高调。在胚珠体外培养的培养液中添加果胶前体核苷糖UDP-阿拉伯糖和UDP-半乳糖醛酸能显著速进纤维的伸长,而加入半纤维素前体核苷糖UDP-木糖对纤维伸长没有影响。实时定量PCR结果显示乙烯能够诱导GAE1和UXE1的表达。综合分析表明,果胶前体核苷糖的生物合成对棉纤维细伸长非常重要,并且受乙烯信号的调控。更多还原

【Abstract】 Cotton fibers are single-celled trichomes differentiated from outer integuments of the ovule. As the most prevalent natural raw materials used in the textile industry, cotton fibers plays a significant role in the global economy. Also, fiber serves as an excellent single-celled model for studying fundamental biological proc esses, such as cell elongation and differentiation Plant primary cell walls consist of mainly pectin, hemicellulose and cellulose, and are deposited during the cell expansion phase. Here, upon 2-DE separation and MALDI-TOF MS analyses, we identified 93 of 101 protein spots that were preferentially accumulated in wild-type cotton ovules,encoded by 66 genes, from wild-type cotton samples. We subjected this dataset to KOBAS and found that cell wall polysaccharide biosynthesis was the most significantly up-regulated bioc hemical pathway in wild-type samples. Proteomic data of 7 protein spots potentially related to nucleotide sugar inter-conversions identified further by Nano-LC-FTICR-MS. The same set of enzymes were accumulated when presence of 0.1μM ethylene, with their transcripts increased after the treatment. Exogenous lignoceric acid (C24:0) showed a similar effect on protein accumulation and gene activation, indicating that these two compounds may indeed promote fiber elongation by sequentially modulating the production of specific cell wall polymers. GC/MS analysis of sugar compositions from non-cellulose wall polysaccharide fractions revealed that fiber primary cell walls contained significantly higher amounts of arabinose, rhamnose and galacturonic acids (GalA) whereas more xylose and glucose were found in ovule samples.When applied in ovule culture media, UDP-rhamnose, precursor of pectic polymer, was able to stimulate significant fiber elongation, while rhamnose was inactive in the same growth assay. The short-root-hair phenotype of Arabidopsis uer1 knock-out mutant was genetically complemented by the cotton UER1 cDNA. Exogenous UDP-rhamnose, not free rhamnose, produced a similar effect when it was included in MS media for Arabidopsis culture. Molecular studies revealed that, in cut1 and ein2-5 Arabidopsis mutants that are known to involve in biosynthesis of C24:0 and ethylene signaling, respectively, the amount of AtUER1 transcripts was significantly reduced. The short root hairs found in the mutants were also rescued only by exogenous UDP-rhamnose, suggesting that C24:0 and ethylene may promote cotton fiber and Arabidopsis root hair growth by upregulating UER1 expression required for production of pectic polymers. Proteomic, metabolomic and genetic studies indicate that biosynthesis of UDP-Rhamnose is important for cotton fiber and Arabidopsis root hair elongationFurther analysis indicate that GAE1 and UXE1 required for biosynthesis of UDP-arabinose and UDP-GalA were preferentially accumulated in cotton fiber cell. When applied in ovule culture media, UDP-arabinose and UDP-GalA, precursors of pectic polymers, were able to stimulate significant fiber elongation, while UDP-xylose, a hemicellulose precursor was inactive in the same growth assay. GAE1 and UXE1 transcripts were increased when 1 dpa wild-type ovules were cultured in the presence of ethylene. Our results indicate that biosynthesis of UDP-sugars that are precursors of pectic polymers is important for cotton fiber elongation.更多还原