【作者】 余克琴；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 果实发育是一个高度协调的复杂生物学过程。作为非呼吸跃变型果实,柑橘果实发育和成熟过程的研究尽管已经逐步得到重视,但是对其分子调控机制的诠释还远远不够。果实成熟过程中形成的糖、酸和类胡萝卜素等物质是柑橘果实品质的重要组成部分,因此,研究柑橘果实成熟的分子以及类胡萝卜素等的积累机制对提高果实品质具有重要的理论价值。突变体‘红暗柳’甜橙果实在具有红肉性状的同时也表现为高蔗糖和低柠檬酸水平,为我们研究果实品质性状形成的分子机理提供了理想材料。本研究以突变体‘红暗柳’与野生型‘暗柳’为材料,采用表达谱测序技术分析了两者果实发育过程中转录组的差异,并重点描述了甜橙果实发育和成熟过程中的转录组动态变化,同时对一个候选基因CsASR的功能进行分析。主要研究结果如下：1利用Illumina测序平台对‘红暗柳’与‘暗柳’各4个发育时期(120DAF,150DAF,190DAF和220DAF)果实的果肉进行数字化表达谱测序。获得的平均标签数目约4.01M。将测序所得所有标签与参考数据库中的序列比对,发现占clean reads68.1%-76.2%共46,328-76,424条标签与参考数据库中已知功能或未知功能的序列具有同源性。以‘暗柳’为模型研究甜橙果实发育与成熟过程中的转录组变化。总共检测到18,829个基因至少在一个时期表达,其中8,825个基因在所有四个时期都有表达。利用Cluster软件对‘暗柳’甜橙果实发育和成熟过程中基因的表达模式进行聚类分析,所有的18,829个基因被聚成22类。聚类分析的结果也显示在‘暗柳’甜橙里检测到89.7%的基因表达量在果实发育和成熟的过程中发生了变化。比较突变体与野生型的基因表达模式发现两个品种中有20类表达模式是共同的。比较‘暗柳’甜橙不同发育时期转录组分别发现9,377个基因在花后120d和150d差异表达；7,886个基因在花后150d和190d差异表达；7,757个基因在花后190d和220d差异表达。生物学过程的GO注释分类结果显示,这些基因中36.7%被注释18条GO类型,且绝大部分基因被注释到代谢过程、细胞过程、建立定位、生物学调节、着色和逆境响应等。这些差异表达基因主要参与细胞壁代谢和软化、蔗糖代谢、三羧酸循环、类胡萝卜素代谢和逆境响应。比较不同发育时期‘暗柳’‘红暗柳’果实的转录组,结果显示分别有634、568、540和616个基因的表达在突变体和野生型花后120d、150d、190d和220d有显著差异(p<0.05和|log2Ratio|≥1)。许多差异表达基因编码逆境相关蛋白。对差异表达基因的数目进行分析发现在所有研究的这四个时期,突变体上调表达基因数目都少于该时期下调表达基因数目。去除冗余,在‘暗柳’和‘红暗柳’中差异表达的基因数目仅883个。对这些差异表达基因进行聚类分析,发现除花后150d外,与野生型相比超过一半的基因(492)在突变体所有的发育时期均上调表达。仅5个基因被发现在所有四个时期均差异表达。基于分子功能的GO注释分类分析显示883个差异表达基因大多数被注释到蛋白结合、水解酶活性、转移酶活性和转运子活性。GO富集分析显示类胡萝卜素代谢过程和辣椒红素/辣椒玉红素合成酶活性在突变体花后150d的果实显著富集。利用Real-Time PCR的方法对选择的22个差异表达基因在突变体和野生型果实发育和成熟过程中的表达进行验证,获得的基因表达模式与高通量测序所得的结果基本一致,两者的相关系数为0.8379。我们也测定了‘暗柳’和‘红暗柳’果实发育和成熟过程中果肉可溶性糖、有机酸、类胡萝卜素和H202的含量变化。在果实发育和成熟的后期可溶性糖的含量在‘暗柳’和‘红暗柳’中均显著增加。在整个果实发育和成熟过程中,‘红暗柳’果肉中的蔗糖含量均显著高于‘暗柳’,但是柠檬酸含量均显著低于‘暗柳’。总类胡萝卜素和番茄红素在‘红暗柳’中大量积累,但是在‘暗柳’中均维持比较低的含量。随着果实的发育和成熟,H202的含量下降,但是在花后120d,‘红暗柳’果肉中H2O2含量显著高于‘暗柳’。2转录组分析的结果以及前人的报道都显示ASR基因在‘红暗柳’和‘暗柳’中表达差异显著。根据获得的基因片段(FE659120)利用Tail-PCR扩增获得甜橙ASR基因的cDNA全长,命名为CsASR, GenBank登录号为HQ398364。进一步在甜橙中扩增获得DNA全长1213bp,含一个内含子。CsASR的cDNA全长为929bp,开放读码框为540bp,编码179个氨基酸。同源性分析表明甜橙CsASR推导的氨基酸序列与其他物种克隆的ASR基因有较高的同源性。保守区域分析表明CsASR与其他已知的ASR蛋白一样,它们的氨基酸序列都包含两个高度保守的区域。功能预测推测其可能参与转录调节,生长发育,转录和信号转导,概率分别为0.241,0.110,0.072和0.063。Southern杂交分析显示在大多数柑橘品种中,CsASR属于一个小的多基因家族。亚细胞定位分析发现CsASR与其他大多数转录因字一样定位在细胞核中。对CsASR基因在‘暗柳’和‘红暗柳’不同发育时期果实以及不同组织中的表达进行分析,结果发现CsASR基因主要在成熟组织,特别在成熟果实中表达。CsASR基因在‘暗柳’的果肉和果皮中的表达模式相同,但是在‘红暗柳’的果肉和果皮中CsASR的表达模式不同,除了在花后120d,整个发育时期CsASR基因在‘红暗柳’中的表达都比在‘暗柳’中的高。对CsASR基因在不同逆境条件下甜橙愈伤中的表达进行分析,结果显示高盐、低温、热和ABA处理条件下,CsASR基因均被显著诱导表达。用外源ABA处理‘暗柳’和‘红暗柳’转色前果实,结果显示ABA诱导‘红暗柳’果肉中可溶性糖和有机酸含量增加,而在‘暗柳’中,ABA处理导致可溶性糖和有机酸含量降低。但是对于类胡萝卜素我们只检测到花药黄质和α-胡萝卜素,尽管ABA处理后‘暗柳’果肉中类胡萝卜素的含量降低,但是在‘红暗柳’果肉中ABA处理前后类胡萝卜素的含量没有显著的变化。利用农杆菌介导遗传转化在番茄中超量表达CsASR基因,共获得11株抗性植株,T1代分离的阳性植株成熟果实显示与对照不同的红色。这些抗性植株与对照相比在形态学及果实的成熟期上并没有明显的差异。微分干涉显微观察的结果显示超量表达CsASR的番茄成熟果实的果皮细胞呈现红色,而对照为黄色。冷冻切片在明场下也显示两者的细胞结构有所不同,转基因番茄果实的细胞壁与对照相比增厚；进一步在荧光下观察发现转基因番茄果实的细胞壁呈现很强的蓝色荧光,即木质素含量较高,而对照表现不明显。HPLC分析结果显示紫黄质、p-胡萝卜素、番茄红素和八氢番茄红素的含量在CsASR超量表达番茄植株的成熟果实中都显著增加,而叶黄素的含量没有明显的差异。与对照相比,所有检测的类胡萝卜素代谢相关基因在CsASR超量表达番茄植株的成熟果实中都上调表达。GC-MS分析结果显示很多主要代谢产物的含量在转基因番茄果实中均发生了改变。几乎所有检测到的糖类代谢物及包括三羧酸循环代谢中间产物在内的所有检测到的有机酸的含量在CsASR超量表达的番茄果实中都低于对照。很多氨基酸的含量也发生了变化。对CsASR超量表达的番茄果实中主要代谢相关基因的表达分析结果发现主要代谢产物的变化与相应基因表达是吻合的。LC-MS技术检测了转基因番茄果实中ABA的含量,结果发现CsASR超量表达的番茄果实中ABA的含量显著低于对照。表达谱测序结果显示,与对照相比总共有401个基因在CsASR超量表达番茄果实中显著差异表达(FDR<0.001and|log2Ratio|≥1),其中180个基因上调,221个基因下调表达。这些差异表达基因包括9-顺式环氧类胡萝卜素双加氧酶基因、Ring-finger蛋白以及含有APETALA2(AP2)结构域的蛋白等。利用KEGG数据库对RNA-seq的结果进行分析,结果发现主要代谢过程、次生代谢过程、植物激素信号传导和类胡萝卜素生物合成等代谢途径在CsASR超量表达番茄果实中发生显著变化。更多还原

【Abstract】 Fruit ripening is a highly coordinated and complicated biological process. Great importance has been gradually attached to the study on the fruit development and ripening of the non-climacteric fruit-citrus. However, an interpretation of the molecular mechanism of fruit development is far from enough. Sugars, organic acids, and carotenoids attained during fruit ripening are major components of citrus fruit quality. It was very important to study the molecular mechanism regulating fruit ripening and carotenoid accumulation for the improvement of citrus fruit quality. The red-fleshed mutant’Hong Anliu’, characterized as high sucrose and low citric acid, was ideal for the study on the molecular mechanism of the formation of fruit quality straits. This study provided a description of the transcriptomic changes occurring during fruit development and ripening in sweet orange, along with a dynamic view of the gene expression differences between the wild type ’Anliu’(WT) and the mutant ’Hong Anliu’(MT). An important candidate gene CsASR was functionally characterized. The main results were as follows:1The WT and MT fruit pulp harvested at120,150,190, and220days after flowering (DAF) was subjected to RNA-seq using an Illumina sequencing platform. The average number of tags produced for each library was4.01million. Mapping tags to a reference citrus unigene dataset identified between68.1%and76.2%of the tags (46,328-76,424) were homologous to sequences with known or unknown function.We solely used WT as a model to demonstrate the transcriptome changes during fruit development and ripening. A total of18,829genes were detected in at least one of the four stages in WT, of which8,825genes were expressed in all the four stages. The cluster analysis of gene expression patterns during fruit development and ripening arranged18,829genes into22groups. It also revealed that the abundance of89.7%of the transcripts detected in the WT pulp varied over the course of fruit development and ripening. A comparison of expression patterns between WT and MT revealed that20of the groups were common to both.A comparison of the transcriptomes of different developmental stages in WT identified9,377,7,886, and7,757were differentially expressed between120DAF and150DAF,150DAF and190DAF, and190DAF and220DAF, respectively. Of these,36.7%were assigned to one of18GO categories. The categories "metabolic process","cellular process","establishment of localization","localization","biological regulation","pigmentation", and "response to stimulus" based on biological process captured most of these genes. Many genes were associated with cell wall metabolism and softening, sucrose metabolism, the TCA cycle, carotenoid biosynthesis, and stress response.The comparison between the transcriptomes of WT and MT identified634,568,540, and616genes were significantly differentially expressed at p<0.05and|log2Ratio|>1in the four developmental stages, respectively. Many encode stress-related products. At all the four developmental stages the number of up-regulated genes was less than that of down-regulated genes. Only883genes were differentially expressed between WT and MT after the removal of the redundant. The cluster analysis of these genes showed over one half (492) turned out to be up-regulated in MT at all the developmental stages except150DAF. Only five genes were detected as differentially expressed at all four stages. The GO categories of differentially expressed genes based on the molecular function revealed that most encoded products associated with "protein binding","hydrolase activity","transferase activity" and "transporter activity". GO enrichment analysis revealed carotenoid metabolic process and capsanthin/capsorubin synthase activity were enriched at150DAF in MT.Q-Real-Time PCR validation of the transcription profiles for22of the differentially expressed genes indicated a good correlation between transcript abundance assayed by real-time PCR and RNA-seq data, with an overall correlation coefficient of0.8379.The dynamics of pulp soluble sugar, organic acid, carotenoid and H2O2content were monitored during fruit development and ripening in WT and MT. The content of soluble sugars increased markedly during the late stages of fruit development and ripening in both WT and MT. The concentration of sucrose was higher but the citris acid content was much lower in MT than in WT throughout fruit development and ripening. Carotenoids and lycopene both accumulated over time in MT, but remained at a low level in WT. H2O2content fell as the fruit developed and ripened, but was higher in MT than in WT at120DAF.2Our transcriptomic analysis and previous reports indicated ASR gene was significantly differentially expressed. The full-length cDNA of sweet orange ASR, designated as CsASR, was cloned based on the EST sequence (FE659120) and deposited in Genbank (accession number HQ398364). CsASR cDNA was929bp long and contained an open reading frame of540bp. The deduced protein contained179amino acids. Sequence homology analysis of amino acid showed CsASR shared considerable identity with other ASR proteins from various plant species. CsASR contained two highly conserved regions as other ASRs. The function prediction suggested that the CsASR protein might be involved in transcription regulation (0.241), growth factor (0.110), transcription (0.072), and signal transducer (0.063).Southern blot analysis suggested CsASR belonged to a small multi-gene family in most citrus species. Subcellular localization analysis revealed CsASR was localized in the cell nucleus.CsASR mRNA accumulation was detected in various tissues and fruits during fruit development and ripening. The result showed CsASR was mainly expressed in mature tissues, especially in mature fruits. The expression pattern of CsASR was the same in the pulp and peel of WT, but different in the pulp and peel of MT. CsASR transcription level was higher in’Hong Anliu’ than in’Anliu’ throughout fruit development except at120DAF. CsASR was significantly induced under cold, heat and salt stress, and ABA treatment.Exogenous ABA was applied to the pulp of WT and MT before colour break. All detected soluble sugars and organic acid content was reduced in WT fruit pulp but increased in MT fruit pulp after ABA treatment. We just detected two carotenoid compositions:Antheraxanthin and a-carotene in treated pulp. Although the content of carotenoid was lower in ABA treated WT pulp than that in the control, there is no difference in MT after treatment.We overexpressed CsASR in tomato via Agrobacterium tumefaciens-mediated transformation, and eleven independent transgenic lines were obtained. The mature fruit of the T1segregation developed to a different red colour, however, there was not other visual significant difference in phenotype and fruit maturity compared to the wild type. Differential interference microscope results showed that pericarp cells of CsASR overexpression tomato fruits appeared red, while the control was yellow. Frozen section watching under a bright field revealed the cell structure of transgenic tomato fruits was different from that in the wild type, and the cell wall of the former was thickening; further the cell wall of the transgenic tomato fruits presents strong blue fluorescence under fluorescence, suggesting the lignin content is high. While, ittle blue fluorescence was observed in the control.HPLC analysis revealed the content of violaxanthin, β-carotene, lycopene and phytoene all increased significantly in CsASR overexpressed fruits. No difference in the lutein content was observed. The expression level of all detected carotenoid metabolism-related genes was up-regulated in transgenic fruits compared to the wild type.GC-MS profiles revealed that the CsASR overexpression fruits displayed substantial changes in the level of primary metabolites. The content of almost all detected sugars and organic acids including the tricarboxylic acid cycle intermediates was lower in the CsASR overexpression fruits than in the wild type. Many amino acids content was also altered. Analysis of the expression profiles of related genes suggested the changes of expression levels of these genes were in agreement with the observed levels of metabolites. The result of LC-MS showed CsASR overexpression fruits had lower ABA level than that in the wild type.The transcriptome sequencing data showed401genes were significantly differentially expressed (FDR≤0.001and|log2Ratio|≥1) in CsASR overexpression fruits compared to the wild type. Of these180genes were up-regulated and221genes down-regulated in transgenic fruits, including NCED gene, APETALA2(AP2) domain-containing proteins, and ring-finger domain-containing protein. Analysis of the RNA-seq data using KEGG database revealed primary metabolism, biosynthesis of secondary metabolites, plant hormone signal transduction, and carotenoid biosynthesis were significantly changed in transgenic fruits.更多还原