【作者】 韦艳萍；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2019， 博士

【摘要】 白菜作物(Brassica rapa,2n=20,AA),是十字花科芸薹属“禹氏三角”中重要经济栽培作物之一,分布地域广,形态多样性丰富。白菜生长发育过程中,如种子的萌发,低温春化诱导的开花形成,花粉的发育,叶球的形成等与生产相关的重要农艺性状都涉及光合产物蔗糖的合成、转运、分配以及利用。并且,蔗糖代谢形成的可溶性糖,不仅作为白菜类作物重要物质影响其口感风味,而且糖代谢中间产物也作为其他营养物质的合成成分,如芥子油苷的前体物质参与芥子油苷的合成,或与黄酮类物质结合参与白菜花器官蜜源标记引诱昆虫帮助授粉。再者,蔗糖代谢通过其代谢本身形成的渗透物质或者相关的糖信号,参与各种生物胁迫和非生物胁迫响应以增强环境的适应性。白菜叶球的形成,因其增加了叶菜类蔬菜的可食性,储藏性和商品性,常作为育种工作者首先关心的农艺性状。目前,白菜叶球形成的调控机制的研究已经从不同层面展开,如叶球形成的组织学、生理学、几何结构、遗传规律、植物激素以及光照强度等。蔗糖代谢,尽管其作为最基本的代谢路径,涉及光合产物在白菜结球初期不同叶片和细胞中糖分的重新分配引起叶片不均匀生长,促使叶球形成,其相关的分子机制并不清楚。而且,近年来,因蔗糖代谢及其信号转导途径涉及的调控网络复杂且庞大,白菜作物关于蔗糖的研究主要围绕在相关可溶性糖的测量,对其调控网略的分析和挖掘鲜有深入。因此,为了解白菜中蔗糖代谢参与白菜叶球形成的调控机制,在本研究中,首先通过生物信息学方法分析了涉及蔗糖转运、分配和利用的关键成员SWEETs,SUTs和INVs家族基因在不同植物分类群中的扩张分布,功能分化和在十字花科植物中的进化特征。其次,对白菜基因组中鉴定的SWEETs,SUTs和INVs家族基因的复制模式,序列特征和表达情况进行了分析,以明确其在白菜基因组中的基本特性。然后,通过对同一白菜结球品种在结球初期不同温度处理获得的结球和不结球表型,基于RNA-Seq转录组学结合iTRAQ定量蛋白质组学的方法,对低温诱导白菜结球性状涉及蔗糖代谢的调控网络进行解析。主要研究结果如下:1.通过分析蔗糖代谢关键成员SWEETs,SUTs和INVs在植物中的分布,十字花科植物中的进化以及伴随白菜全基因组三倍化后基因的扩张和保留成员的表达模式,结果发现,在不同分类的92种已测序植物中,具有蔗糖转运活性的SWEETs最初来自维管植物,而SUTs不同分类在植物中具有独立的进化方式,INVs中的CWINs出现在裸子植物之后。十字花科中这三个成员都有不同程度的扩增,但都表现在Lineage Ⅰ相对保守,Lineage Ⅲ有大量的丢失。进一步,对白菜基因组中SWEETs,SUTs和INVs的鉴定、聚类、保守结构域和表达分析发现,部分扩张的成员因结构的缺失而不具有转录活性,叶片中转录的旁系同源基因对不同非生物胁迫表达响应具有差异性。综上结果,提供了蔗糖代谢途径SWEETs,SUTs和INVs在白菜基因组中的基本信息,以期为后续解析糖代谢中蔗糖的转运、分配和利用,以及在参与白菜生长发育、逆境胁迫和品质调控等的复杂网络上奠定基础。2.对同一白菜结球品种,在结球初期通过不同的温度处理获得结球表型和不结球表型。基于联合使用RNA-Seq转录组测序和iTRAQ蛋白质组定量技术,对涉及白菜结球的分子动态进行解析。结果表明,在结球表型和不结球表型中,共鉴定出表达丰度显著差异变化的转录本2931个,蛋白质365个。转录本到蛋白质之间的丰度相关性分析发现,所鉴定的转录本和可定量的蛋白质具有很低的相关性(相关系数R=0.1914),但表达丰度具有显著差异变化的成员相关性较高(表达趋势相同的相关系数R=0.7179,表达趋势相反的相关系数R=0.5294)。功能富集分析发现,2.9%显著变化的基因(52个),被过于富集到与糖代谢相关的淀粉/蔗糖代谢路径和糖酵解/糖异生路径。进一步分析确定SWEET家族Clade Ⅲ具有蔗糖转运活性的BrSWEET12-MF2,BRSWEET13-LF,BrSWEET14-MF1,BrSWEET14-MF2,BrSWEET15-LF,BrSWEET15-MF1和BrSWEET15-MF2可能通过减低源端蔗糖的长距离运输增加叶片中糖分的积累。此外,BrSWEET1-MF1,BrSWEET1 6-MF1,BrSWEET1 7-MF1和BrSWEET17-MF2,SUTs 家族的 BrSUT1-LF,BrSUT1-LF,BrSUT9-LF,BrSUT9-MF1,INVs 家族的BrCW1N2-MF1,BrCIN6-LF,BrCWIN4-MF2,BrCWIN5-LFb,BrCWIN5-MF1,BrVIN1-MF2可能通过参与蔗糖的转运和降解参与白菜的结球过程。更多还原

【Abstract】 Chinese Cabbage(Brassica rapa,2n=20,AA),as one of the important economically cultivated Brassica crops,belongs to the Cruciferae family with the characteristics of wide geographical distribution,rich morphological diversity.Sucrose metabolism including sugars synthesis,translocation,partitioning and utilization,is vital for growth and development of cabbage crops,such as the process of seed germination,flowering formation induced by vernalization,pollen development,leaf-head formation,as well as other key agricultural traits which relate to economic production and nutritional quality.Moreover,the soluble sugars formed by sucrose metabolism not only affect the cabbage taste but also improve the cold resistance of cabbage crops by changing sugars content and/or sugar singal.For example,a better taste for cabbage leaves by increasing the soluble sugar content and longer freshness for the detached pakchoi plants by slowing the degradation of sucrose and hexose consumption after an appropriate low temperature.Additionally,the intermediates from sugar metabolism are usually as precursors for other important components(such as precursors of glucosinolates)or linked with flavonoids involving in the nectar guide of cabbage flower to attract insects to pollinate.Furthermore,sucrose metabolism response to a variety of biotic and abiotic stresses to improve environmental adaptability through the formation of osmotic substance or sugar signals.The leafy-head formation of Chinese cabbage,since it has better edibility,storage and marketability,is often concerned as the primary agronomic trait by breeding workers.As so far,the research on the regulation mechanism of leaf-head formation has been carried out from different aspects ranging from histology,physiology,geometric structure,genetic law,plant hormones and light intensity,etc.Sucrose metabolism,although as the most basic metabolic pathway,involves the distribution of photosynthetic products in different leaves and cells in the early stage of head formation,causing uneven growth of leaves and promoting the leaf-head formation.The related molecular mechanism is not clear.Moreover,in recent years,the regulatory network involved in sucrose metabolism and its signal transduction pathway is complex and huge.The research on sucrose in cabbage crops mainly focuses on the measurement of related soluble sugars,and the analysis and excavation of its regulation network are rarely deep.Therefore,to understand the regulation mechanism of sucrose metabolism in Chinese cabbage to participate in the formation of leafy head,in this study,the sucrose metabolism key player of SWEETs,SUTs and INVs family genes which involved in sucrose transport,distribution and utilization were firstly analyzed by bioinformatics methods in the different plant taxa and cruciferous groups to explore its evolutionary footprint and functional differentiation.Secondly,the duplication patterns,sequence characteristics and expression pattern of SWEETs,SUTs and INVs family genes in the Chinese cabbage were analyzed to understand their basic information in the Chinese cabbage.Then,by combing RNA-Seq transcriptome with iTRAQ quantitative proteome technology,low-temperature-induced leafy-head formation for two phenotypes from an early stage of heading Chinese cabbage "chifu" was investigated.The key findings are as follows:1.The footprint and functional differentiation of SWEETs,SUTs and INVs in 92 sequenced genomes from different plant taxa were first investigated by homologous.Identification and phylogenetic relationship suggested that SWEETs with sucrose transport activity were originally from vascular plants,each SUT subclassifications had independent evolution patterns,and the newest CWINs subgroup of INVs originally appeared in gymnosperms plants.Further the footprint and phylogenetic relationship analysis for cruciferous genome-sequenced plants shown that gene members were differentially amplified for those three families but all conservative in Lineage I and part of the loss in Lineage III of Cruciferae family.Additionally,analysis for homologous identification,phylogenetic relationship,conserved domain and expression analysis in different tissues for BrSWEETs,BrSUTs,and BrINVs in triplicated B.rapa genome suggested that some of the retained members have missed part of domain or motif,and/or didn’t check any expression in any tissues.In summary,the work reported here provides the groundwork for SWEETs,SUTs and INV in B.rapa crops,which will lead to a more detailed understanding for sucrose involved sugar translocation,partitioning and utilization involved plant growth and development,as well as a better adaptation for the environment,the trait for a global picture of the molecular dynamics.2.By combing RNA-Seq transcriptome with iTRAQ quantitative proteome technology,low-temperature-induced leafy-head formation for two phenotypes from an early stage of heading Chinese cabbage "chifu"was investigated A total of 2931 transcripts and 365 proteins were identified with significantly changed levels in abundance from heading and non-heading Chinese cabbage.The correlation analysis from transcripts to proteins suggested that rare genes related for all identified transcripts(R=0.1914)and quantitative proteins but reluctant good correlation for significantly different changed members(R=0.7179 for the members with same changing trend and R=0.5294 for members with the opposite changing trend)。KEGG functional shown that considerable numbers were enriched to the pathways of starch/sucrose metabolism and Glycolysis/Gluconeogenesis.And further analysis suggested that sucrose related genes of BrSWEET12-MF2,BRSWEET13-LF,BrSWEET14MF1,BrSWEET14-MF2,BrSWEET15-LF,BrSWEET15-MF1 and BrSWEET15-MF2may increase sugar accumulation by the hold-up of the phloem loading process.Besides,BrSWEET1-MF1,BrSWEET16-MF1,BrSWEET17-MFI andBrSWEETI 7-MF2,BrSUTILF,BrSUTl-LF,BrSUT9-LF,BrSUT9-MF1 from SUT family,BrCWIN2-MFl,BrCIN6LF,BrCWIN4-MF2,BrCWIN5-LFb,BrCWIN5-MF1,BrVIN1-MF2 from INV family may respond to the process of head formation for Chinse cabbage by involving in sucrose translocation and degradation.更多还原