【作者】 詹少华；

【导师】 林毅；

【作者基本信息】 安徽农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 本试验主要研究天然棕色棉纤维色素的化学成分、色素的合成地点、棉纤维发育与色素合成之间关系以及棕色棉SRAP分子标记,以期为彩色棉育种提供依据和参考。本文首先系统研究了天然棕色棉纤维色素的溶解性和纯化方法,包括重结晶法、铅盐/铜盐法、纸层析法和硅胶柱层析法,设计了高效率提取棕色棉纤维色素的工艺流程,通过薄层层析等方法证实了高效率纯化方法得到的色素具有足够纯度。利用纯化色素进行化学性质鉴定、波谱测定以及生理学指标分析,推断出天然棕色棉纤维色素为缩合单宁氧化而来的醌类化合物。分析了棕色棉纤维色素的分布规律、种皮和棉纤维中棕色素、缩合单宁变化等。结果表明:（1）棕色棉种皮中色素含量高于棉纤维中色素含量;（2）棕色棉种皮细胞色素加深期早与棉纤维细胞;（3）棕色棉没有发育成纤维的种皮细胞颜色也是深棕色;（4）棕色棉纤维颜色深浅的主要原因是色素在纤维和种皮之间的分配比例不同;（5）棕色棉纤维靠近种皮一端的颜色最深;（6）种皮细胞中的缩合单宁含量高于棉纤维细胞中缩合单宁的含量;（7）在5-30 DPA期间,棕色棉的纤维细胞和种皮细胞中的缩合单宁的含量的动态走势是一致的,白色棉则不具有这一特征;（8）白色棉的种皮中存在着与棕色棉纤维中相同的色素;（9）棕色棉纤维颜色加深最快的时期在棉铃脱水成熟期,此阶段棉纤维细胞代谢的活力正在逐渐下降;（10）如果色素运输到纤维细胞中发生在25 DPA以后,就很难解释在试验中观察到的色素在棉纤维上的分布特征;（11）彩色棉纤维颜色都与种皮颜色一致,棕色棉种皮颜色是棕色,绿色棉种皮颜色是绿色。以此,推断棕色棉纤维色素的合成地点在种皮细胞中。试验结果还表明:（1）各发育时期,每一种棕色棉纤维中或种皮中缩合单宁的含量远高于白色棉,并且棕色棉纤维色素含量越高,棉纤维中或种皮中缩合单宁的含量就越高;（2）各发育时期棕色棉纤维细胞磨碎液滴加1%FeCl₃（g/g）试剂,均表现为蓝绿色,而白色棉不具有这种现象或很微弱;（3）成熟棉纤维的色素含量越高,5-30 DPA期间种皮细胞中的缩合单宁运输到纤维细胞中的效率就越高;（4）成熟棉纤维中色素的含量与20-25 DPA棉纤维中缩合单宁的含量、棉纤维中缩合单宁总平均含量极显著相关。结合化学性质鉴定的结果,说明无色的缩合单宁是棕色棉纤维色素的合成前体。以白色棉泗棉3号为对照,测定了4种棕色棉纤维各发育阶段的生化物质组分变化的规律,结果显示:白色棉40 DPA含水率、10 DPA还原糖高于棕色棉,30-40 DPA还原糖含量反而比棕色棉低,白色棉20 DPA可溶性蛋白质出现高峰期,棕色棉相应的蛋白质高峰期出现在25 DPA,白色棉纤维生长的各阶段纤维素的含量均高于棕色棉;成熟棉纤维色素的含量与10 DPA还原性糖含量、30-40 DPA纤维素含量呈负相关,达到显著水平（p＜0.05）,与35DPA还原性糖含量呈正相关,达到极显著水平（p＜0.01）,与15DPA纤维素含量呈负相关,达到极显著水平（p＜0.01）。比较了5种棉花基因组DNA提取方法,结果表明预处理除酚法可以明显提高棉花基因组DNA的质量。SRAP-PCR反应体系的优化的结果是:25 ml反应体系中加入25 m mol/L Mg²⁺2.0μl-10 m mol/L dNTPs 0.5μl-DNA模板60 ng-5U/μl TaqDNA聚合酶0.2μl-10 mmol/L引物1.5μl。根据SRAP分子标记,在Jaccard相似系数0.88的水平上,可以将本试验的10个棉花品种聚为4类。系统地研究了棕、绿彩色棉棉铃生长发育动态,发现棉铃动态发育过程中,多数指标棕色棉和白色棉相接近,而绿色棉棉铃的动态发育与白色棉相差较大;棉铃长度、铃横径、种仁长度、单铃体积都是铃龄0-10天呈现直线变化,铃龄15天达到或接近最大值;纤维长度,单铃纤维干重铃龄在15天以后继续增长。根据白色棉与棕色棉杂交后代的表现型,分析了天然棕色棉纤维色素的遗传规律。自交F₂代三种类型的数量为:深棕色类型62株、中棕色类型121株和白色类型78株,卡平方检验符合1∶2∶1的比数（p＞0.05）。亲本及杂交后代棉纤维色素含量的定量测定表明,F₂代三种性状呈现不连续的分布,说明天然棕色棉纤维颜色的遗传方式是棕色对白色为不完全显性遗传。更多还原

【Abstract】 The main aim of our study was to research chemical structure,synthetic location and the SRAP molecular marker of the pigment in naturally colored brown cotton fiber.Also the relationships between the pigment synthesis and the cotton fiber development were analyzed.The experiments would contribute to breeding of naturally colored cotton.Solubility and purifying methods of the pigment in naturally colored brown cotton fiber were researched at first,such as the recrystallization method,lead salt/cupric salt method,paper chromatography method and silica gel column chromatography method.A efficient technological process for purifying the pigment was designed,and pureness of the pigment was verified by the thin layer chromatography.The pigment was deduced as quinone from condensed tannin oxidation according to the chemical characteristics,the wave spectra of the pigment and physiological measurements.The pigment distribution regulation on the naturally colored brown cotton fiber,the pigment contents and the dynamic contents of condensed tannin in the cotton fiber or coat seeds were analyzed.The results indicated（1）the pigment contents in coat seeds cells were higher than that of cotton fiber;（2）the time of coat seeds appearing color earlier than that of cotton fiber;（3）the color of coat seeds cells which did not develop to cotton fiber were dark brown too;（4）the color of naturally colored brown cotton fiber was dependent on the ratios of pigment between the cotton fiber and coat seeds cell;（5）the darkest color of the naturally colored brown cotton fiber was near the coat seeds cells;（6）condensed tannin contents in the coat seeds cells were higher than that of cotton fiber;（7）dynamic tendencies of condensed tannin contents in naturally colored brown cotton fiber were conform to that coat seeds during 5-30 DPA,but conventional white cotton had not the property;（8）the pigment in the coat seeds cells was same as the pigment in naturally colored brown cotton fiber;（9）the displaying color speed of the naturally colored brown cotton fiber was fastest at the opening boll stage,while metabolism vitality of the fiber was decreased gradually; （10）the pigment distribution regulation on the naturally colored brown cotton fiber did not support the hypothesis that the pigment was convey to the fiber of naturally colored brown cotton after 25 DPA;（11）the color of naturally colored cotton fiber was same as that of coat seeds,such as coat seeds color of nature brown cotton was brown,and coat seeds color of nature green cotton was green.So we got a conclusion that synthesis location of the pigment in naturally colored cotton fiber was in the coat seeds cells.The results also indicated（1）the condensed tannin of naturally colored brown cotton fiber or coat seeds were higher than that of conventional white cotton,and the more pigment contents in naturally colored cotton fiber,the more condensed tannin contents in naturally colored brown cotton fiber or coat seeds during fiber every development stages;（2） triturating liquid of the naturally colored brown cotton fiber blended with 1%FeCl₃（g/g） presented blue-green color,the phenomenon was due to higher concentration of the condensed tannin in naturally colored brown cotton fiber;（3）the more the pigment content in naturally colored brown fiber,the more the efficiency of the pigment conveying to fiber from coat seeds during 5-30 DPA;（4）the pigment contents in the mature cotton fiber had significant correlation with the contents of condensed tannin in colored cotton fiber during 20-25 DPA and the mean contents of the condensed tannin（p＜0.01）.we claimed the condensed tannin was the precursor of the pigment in naturally colored cotton fiber according to the characteristics and the chemical properties.The biochemical composition in the 4 kinds of natural brown cotton fiber has been determined from the early stages of elongation to the period of secondary wall formation, comparing with normal cotton Simian-3.The measurement revealed that the moisture content at 40 DPA and the reductive sugar at 10 DPA of normal cotton were higher than that of natural brown cotton,but the reductive sugar of the brown cotton fiber from 30 to 40 DPA showed opposite trend.In contrast to the extractable protein peak of white cotton fiber at 20 DPA,the peak of nature brown cotton fiber arisen at 25 DPA.The cellulose contents of normal cotton at every stage during fiber development were higher than natural brown cotton.The pigment content to the amounts of reductive sugar at 10 DPA and cellulose at 30- 40 DPA was negative correlation significantly at the 0.05 level,and to the 35 DPA reductive sugars content positive correlation significantly at the 0.01 level,to the cellulose content at 15 DPA negative correlations significantly at the 0.01 level.5 kinds DNA extraction methods were compared,and the tests indicated pre-removed method improved the quality of DNA evidently.The result of SRAP-PCR optimization was 25 m mol/L Mg²⁺2.0μl,10 m mol/L dNTPs 0.5μl,template DNA 60 ng,5U/ml TaqDNA polymerase 0.2μl,10 mmol/L primer 1.5μl in 25 ml reaction system.According to SRAP molecular marker,10 cotton cultivars divided into 4 kinds at Jaccard similar coefficient 0.88 level.The bolls development characteristics of naturally colored cotton were studied in comparison with Simian3.The results showed the bolls development characteristics of nature green cotton were obviously different from the characteristics of the white cotton, while nature brown cotton were closed to the white cotton.The length,the maximum width, the volume of a single boll and the length of a single seed,have the liner relationship to the boll age during 0-10 days,and get the maximum at the 15th of boll age.The fiber lengths, the dry weight of a single boll continue to increase after the 15th of boll age.The heredity regulation of the pigment in the naturally colored brown cotton fiber was analyzed on the base of filial generation phenotype.According to eyeballing,the phenotype of self cross F₂ generation could be divided into three kinds.The three kinds were dark brown,middle brown and white.The number of dark brown was 62,middle brown was 121,and the white was 78.The ratio of the self cross F₂ generation was in accord with 1:2:1 by Chi-Square Test（p＞0.05）.The contents of the pigment in parent strains and filial generation cotton fiber were measured.The measurement showed the three genetic traits of F₂ generation were discontinuous distribution.So we claimed genetic traits of brown were incomplete dominant inheritance.更多还原