【作者】 刘景安；

【导师】 许勇；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2013， 博士

【摘要】 西瓜果实品质发育是一个十分复杂的动态过程，主要包含果实大小、糖含量与糖分组成、果肉的颜色、质地和风味等一系列的变化。本项研究的主要目的是探明西瓜果实品质发育过程中主要品质物质构成的变化特征、品质形成相关酶活性的变化规律以及相关基因表达情况。本研究选取高糖栽培西瓜品种97103（Citrullus lanatus subsp. vulgaris East-Asia Ecotype）和无糖近缘野生变种PI296341-FR（Citrullus lanatus subsp. lanatus）的几个关键发育阶段的西瓜果实，采用生理生化和高通量测序技术，系统分析了西瓜果实发育中品质形成的生理生化机制及品质相关基因表达模式。得到如下主要结果：1.栽培西瓜品种97103和野生变种PI296341-FR之间果实物质构成差异比较1.1.97103和PI296341-FR果实干物重的显著差异体现了光合同化物卸载和分配的差异。成熟果实中，97103卸载的光合同化物约为PI296341-FR的1.3-1.5倍；97103果实中卸载的光合同化物主要分配到果肉，以可溶性糖的形式积累，PI296341-FR主要分配到种子。1.2.97103和PI296341-FR果肉和果皮间糖分积累存在显著差异。97103果肉积累了大量的果糖、葡萄糖和蔗糖；97103果皮只积累很少量的葡萄糖和果糖，几乎不积累蔗糖。蔗糖的积累是决定97103果实甜度的主要因素。PI296341-FR果肉和果皮糖分的积累很少，以果糖和葡萄糖为主，蔗糖几乎为零。1.3. PI296341-FR果肉果胶和粗纤维含量比97103高3倍以上，是造成两个品种果肉硬度差异的主要原因。97103西瓜果实主要积累苹果酸。97103果肉苹果酸含量比PI296341-FR稍高，这一差异决定了现代栽培西瓜有别于野生西瓜的独特风味。2.栽培西瓜品种97103和野生变种PI296341-FR果实品质差异的生理生化机制2.1..甜组织（97103果肉）酸性a-半乳糖苷酶活性显著高于非甜组织，进一步支持了甜组织比非甜组织（97103果皮、PI296341-FR果皮和果肉）卸载了更多的光合同化物，主要以可溶性糖的形式在甜组织积累。甜组织与非甜组织碱性a-半乳糖苷酶活性几乎无差异。2.2.甜组织发育中后期不可溶性转化酶活性高与非甜组织，是甜组织中卸载光合同化物多于非甜组织的重要因素。可溶性酸性转化酶活性在甜组织发育中后期与非甜组织几乎无差异。甜组织蔗糖合成酶和蔗糖磷酸合成酶显著高于非甜组织，表明甜组织蔗糖的积累与蔗糖合成酶和蔗糖磷酸合成酶密切相关。2.3.97103果实果肉多聚半乳糖醛酸酶（PG）和纤维素酶（Cx）活性明显高于PI296341-FR果肉，是导致果实质地软化的重要酶。苹果酸脱氢酶活性显著差异造成了97103果肉与PI296341-FR果肉苹果酸含量差异。3.栽培品种和野生变种西瓜果实品质形成的基因表达谱3.1选取栽培品种97103发育的四个和野生变种PI296341-FR发育的六个重要时期，开展97103果肉和其果皮及PI296341-FR果肉进行链特异RNA序列分析。共确定了2,452个基因在97103果肉、826个基因在其果皮中及322在PI296341-FR果肉中差异表达。GO term富集分析表明，97103果肉、97103果皮和PI296341-FR果肉果实发育中生物过程如细胞壁构建、防御应答反应均发生了显著变化，而己糖和单糖代谢过程只在97103果肉中显著改变。3.2.在西瓜注释基因组中，共有62个糖代谢酶相关基因。转录组分析确定13个糖代谢酶相关基因果实发育中差异表达。西瓜果实发育的基因表达谱更能在基因表达水平反应糖代谢相关基因表达差异，西瓜果实甜组织比非甜组织有更多的基因差异表达。西瓜果实糖代谢基因表达调控机制复杂，部分相关基因表达与糖代谢酶活性有一致的动态变化趋势，但也有不同。基于西瓜果实糖代谢基因差异表达分析，建立了西瓜果肉细胞糖代谢模型：在西瓜果肉发育中，α-半乳糖苷酶、不可溶性酸性转化酶，蔗糖合成酶，蔗糖磷酸合成酶，UDP-葡萄糖异构酶，可溶性酸性转化酶和UDP半乳糖焦磷酸化酶相关酶基因家族存在一个或多个差异表达基因，这些差异表达的基因可能是调控果实糖卸载和糖代谢及积累的关键基因。更多还原

【Abstract】 Fruit quality development of watermenlon is a complex process, mainly including changes of fruitshape and size, sugar content and composition, flesh color, texture and flavor. In this study, thepurpose is to investigate the variation of substances and enzymes that related to fruit quality, geneexpression profiling of fruit quality formation during watermelon fruit development. East-Asia Ecotypewatermelon accession97103(Citrullus lanatus subsp. vulgaris East-Asia Ecotype) with high sugarcontent and the wide type watermelon accession PI296341-FR（Citrullus lanatus subsp. lanatus）withvery low sugar content were used to study the physiological and biochemical mechanism and geneexpression profiling in several critical stages of fruit quality formation during watermelon fruitdevelopment via physiological and biochemical analysis and high-throughput sequencing technology.The main results are as following:1. The comparison of physical differences between97103with high sugar content andPI296341-FR fruit with low sugar content1.1. The significant difference of dry matter content between97103and PI296341-FR fruitindicated the differences of photosynthates unloading and partitioning. Unloaded photosynthates in97103matured fruit was about1.3-1.5times higher than that in PI296341-FR fruit. More unloadedphotosynthates were mainly stored in97103flesh tissue in the form of soluble sugars. In comparison,less photosynthates were unloaded into PI296341-FR fruit and mainly stored in seeds.1.2. The contents of sucrose, fructose and glucose in97103central flesh were high, while thecontent of sucrose, fructose and glucose were very low in97103mesocarp, PI296341-FR central fleshand mesocarp. The accumulation of sucrose was the key factor determining the final sweetness of97103fruit flesh.1.3. The lower content of pectin, crude fiber and malate in97103fruit flesh than that inPI296341-FR lead to the attractive agricultural traits of cultivated watermelon, including crispy textureand distinct flavor.2. The physiological and biochemical differences for watermelon fruit between97103andPI296341-FR2.1. In comparison, the enzyme activity of acid alfa-galactosidase in sweet tissue was significanthigher than in non-sweet tissues. It indicated that more photosynthates were unloaded into sweet tissuethan non-sweet tissues, and accumulated in the form of soluble sugars in sweet tissue. There was almostno difference in the enzyme activity of alkaline alfa-galactosidase between sweet tissue (97103centralflesh) and non-sweet tissue (97103mesocarp, PI296341-FR flesh and mesocarp PI29634).2.2. The enzyme activity of Insoluble acid invertase (IAI) were higher in sweet tissue than innon-sweet tissues.It indicated that more photosynthates were unloaded into sweet tissue than non sweettissues and IAI played an important role in photosynthates unloading. No difference was observed inthe enzyme activity of soluble acid invertase between sweet tissue and non-sweet tissues in the latter stage of fruit development. The enzyme activities of sucrose synthase (SuSy) and sucrose phosphatesynthase (SPS) were significant higher in sweet tissue than those in non-sweet tissues. These resultsindicated that sucrose accumulation was closely related to the enzyme activities of SuSy and SPS.2.3. The enzyme activities of polygalacturonase (PG) and cellulose (Cx) in97193fruit flesh weresignificant higher than in other tissues including97103mesocarp, PI296341-FR flesh and mesocarp.These results indicated that the two enzymes should be involved in fruit texture solfting. The significantdifference of malate content between97193fruit flesh and PI296341-FR flesh may be due to theenzyme activity difference of malate dehydrogenase (MDH).3. The comparison of gene expression profiling between cultivated97103and wild typePI296341-FR during fruit quality developmentFour critical stages for97103and six critical stages for PI296341-FR had been chosen forstrand-specific RNA-seq analysis in97103flesh,97103mesocarp and PI296341-FR flesh during fruitdevelopment.2,452,826and322genes were identified that were differentially expressed in97103flesh,97103mesocarp and PI296341-FR flesh, respectively, during fruit development. GO termenrichment analysis indicated that, during fruit development, biological processes such as cell wallbiogenesis, biogenesis and defense responses were significantly altered in97103flesh,97103mesocarpand PI296341-FR flesh, whereas hexose and monosaccharide metabolic processes were onlysignificantly altered in97103flesh, supporting sugar contents observed among three tissues.The annotated watermelon genome contains a total of62sugar metabolic enzyme genes. Ourtranscriptome analysis identified13sugar metabolic genes that are differentially expressed during97103flesh development and among three tissues. The differences of differentially expressed sugarmetabolic genes can be better supported in transcriptome level and more genes were different expressedin sweet tissue than in non-sweet tissues of watermelon fruit. Some differentially expressed sugarmetabolic genes correlated with activities of sugar metabolic enzymes and some not, possible reasonwas the mechanism for sugar metabolic processes were complex.Based on the analyses of differentially expressed sugar metabolic genes, we propose a model forsugar metabolism in cells of watermelon fruit flesh: During watermelon flesh development,α-galactosidase (AGA), insoluble acid invertase (IAI), sucrose phosphate synthase（SuSy）, sucrosephosphate synthase (SPS), UDP-glucose4-epimerase (UGE), soluble acid invertase (SAI) andUDP-galactose pyrophosphorylase (UGGP) had one or more expressed genes in enzyme families, andthese expressed genes involved in regulating sugar unloading and metabolism as key genes.更多还原