【作者】 高英；

【导师】 陆建良；

【作者基本信息】 浙江大学 ， 茶学， 2019， 博士

【摘要】 茶树组织培养是珍稀种质资源保存和扩繁的基本手段之一,也是基因功能研究和转基因的重要工作基础。迄今,茶树再生体系尚不完善,其原因主要与外植体脱分化、再分化调控机制不明晰有关。本论文利用转录组和sRNA测序、qPCR和MASP等技术研究了茶树茎段和叶片外植体脱分化、再分化过程中功能基因和miRNA差异表达以及基因组DNA甲基化变化规律,以期明确茶树组织脱分化和再分化表观遗传调控机制。获得的主要结果如下:(1)转录组分析和qPCR验证结果显示,“植物激素信号转导”、“玉米素生物合成”、“DNA复制”、“谷胱甘肽代谢”、“光合作用”和“次级代谢相关途径”等通路的调整与茶树组织脱分化、再分化关系密切,其中ARR5、GH3.1、IAA18、IAA29、CYCD3-1 CDKB2-2、MYB15、ARF18和ERFR P2-12等生长素/细胞分裂素相关基因对激素信号转导途径的调节作用显著。特化组织(如茎段、叶片、根和芽)与非特化组织(愈伤初期、愈伤)状态差异实质上是众多基因表达模式的不同。愈伤形成与叶绿体退化以及乙烯和细胞分裂素/生长素代谢诱发的细胞增殖密切相关;根的形成与离子转运、极性生长、质体重建等有关;芽的分化则与蛋白加工、光合作用、次生代谢及氧化胁迫缓冲等有关。(2)sRNA测序结果显示,茶树茎段外植体、愈伤、再分化根和芽等材料中检测到204个miRNA,各样品间表达量差异2倍以上的有177个。经qPCR验证,csn-miR167d等12个miRNA与茶树组织脱分化及再分化过程关系密切。miRNA-靶基因关联分析显示,csn-miR167D、csn-miR156、csn-miR166a-3p、csn-miR396、csn-miR157d-5p和csn-miR393c-3p分别通过调节相应的ERF3、SPB1、ATHB 15、AIP15A、GST和ATG18B等靶基因的表达活性,进而影响茶树脱分化和再分化进程。(3)MSAP分析显示,茎段和叶片外植体甲基化水平较高,超过45%;愈伤形成时甲基化水平逐渐降低;茎段愈伤生根与叶片愈伤生根DNA甲基化水平较为接近,分别为35%和34%。经甲基化多态性位点回收,获得了 37条长度在34-334bp之间可读序列,其中30余条序列与茶树、番茄和野牵牛等植物核基因组有较高同源性。亚硫酸氢钠脱氨处理结果显示,甲基化特异性条带中,除了酶切位点的甲基化外,序列内部还存在大量胞嘧啶甲基化位点,而且这些位点的甲基化状态在脱分化和再分化过程中存在动态变化。但甲基化特异性位点的变化与相关基因表达以及脱分化和再分化表型等关系尚需深入研究。更多还原

【Abstract】 Tissue culture is one of the basic means for preservation and propagation of rare germplasm resources,and also an important foundation for gene function and transgenic research.Up to now,the regeneration system of tea plant is far from perfection,which is mainly caused by unclear regulation mechanism of dedifferentiation and redifferentiation.In order to elucidate the epigenetic regulation mechanism of tissue dedifferentiation and redifferentiation in tea plants,the differential expression of functional genes and microRNAs and the methylation of genomic DNA during the dedifferentiation and redifferentiation processes of stem and leaf explants were studied through transcriptome and sRNA sequencing,qPCR and MASP.The main results obtained are as follows:(1)The results of transcriptome analysis and qPCR validation showed that the regulation of’plant hormone signal transduction’,’zeatin biosynthesis’,’DNA replication’,’glutathione metabolism’,’photosynthesis’ and ’secondary metabolic pathways’,were closely related to the dedifferentiation and redifferentiation of tea plant tissues.Auxin/cytokinin-related genes such as ARR5,GH3.1,IAA18,IAA29,CYCD3-1,CDKB2-2,MYB15,ARF18 and ERF RAP2-12,played significant roles in regulating hormone signaling pathways.The difference between the status of specialized tissues(such as stem segments,leaves,roots and shoots)and non-specialized tissues(primarily-callus,callus)might be determined by the differential expression patterns of relevant genes.Callus formation might be closely related to chloroplast degradation and cell proliferation induced by ethylene and cytokinin/auxin metabolism;root redifferentiation might be associated with ion transportation,polar growth,plastid remodeling;protein processing,photosynthesis,secondary metabolism and oxidative stress buffering might be responsible for bud inducing.(2)The results of sRNA sequencing showed that 204 microRNAs were detected in stem explants,callus as well as redifferentiated roots and shoots,while more than two-fold change of the 177 microRNAs expression was observed among the different samples.qPCR confirmed that 12 microRNAs(such as csn-miRNA-167d)were closely related to the dedifferentiation and redifferentiation of tissue culture in tea plants.Association analysis of miRNAs and their targeted genes showed that csn-miR167d,csn-miR156,csn-miR166a-3p,csn-miR396,csn-miR157d-5p and csn-miR393c-3p would significantly affect the dedifferentiation and redifferentiation through regulating the expression of the relevant targeted gene ERF3,SPB1,ATHB 15,AIP 15A,GST and ATG18b respectively.(3)MSAP analysis showed that the DNA methylation level of stem and leaf explants was higher than 45%,and that the level gradually decreased during callus formation.Similar methylation levels were observed in the stem callus-derived(35%)and leaf callus-derived roots(34%).Thirty-seven readable sequences with 34-334bp in length were obtained by recovering the methylation polymorphic loci,and more than 30 of them were highly homologous to the nuclear genomes of tea pant,tomato and Ipomoea indica.The sequencing results after treating with sodium bisulfite showed that a large number of cytosine methylation sites were achieved in the methylation-specific bands besides the restriction endonuclease recognition site,and the methylation status of these sites changed dynamically during the dedifferentiation and redifferentiation of the tea plant.However,the relationship among the changes of methylation specific sites,the expression of related genes as well as dedifferentiation and redifferentiation phenotypes needs to be further studied.更多还原