【作者】 双叶；

【导师】 红艳；

【作者基本信息】 内蒙古民族大学 ， 中药学， 2025， 硕士

【摘要】 目的:北沙参为伞形科珊瑚菜的干燥根,以种子繁殖为主,但种子具有深度休眠特性,给生产带来了诸多不便。目前关于休眠的研究主要集中在解除休眠条件方面,对有关其生理变化、基因表达等研究尚不明确。系统探究北沙参种子休眠的生理生化变化及转录组技术筛选与植物激素信号通路相关的基因,揭示这些基因在休眠解除过程中的动态表达模式及其功能,旨在确定北沙参种子的基因表达使生理方面做出响应进而解除休眠,丰富种子休眠调控理论,为深入研究分子机制奠定基础。方法:本研究以北沙参种子为材料,通过动态监测北沙参种子的形态特征,酶活性及内源激素的变化,阐明北沙参种子解除休眠过程中的各生理特性指标的变化规律,结合转录组学分析多个植物激素代谢关键基因和种子休眠解除与萌发相关基因的表达情况,在分子层面对北沙参种子休眠与萌发进行解释,并发掘北沙参种子休眠的主要效应基因,阐明北沙参种子的休眠机理。结果:（1）北沙参种子在5℃条件下进行裸层积处理,层积14天后开始迅速萌发,至42天时发芽率达到峰值（100%）,同时发芽指数也达到最高值,平均发芽时间显著缩短。在层积过程中,种胚的发育呈现持续增长趋势。层积初期（0天）时,种胚较小,胚长为1.0～1.03mm,胚率为13.33%;随后,种胚在0～42天内快速生长,并在45天时种胚已突出种皮,胚长增至4～5mm,胚率达到最大值39.67%。（2）随着层积时间的延长,北沙参种子中淀粉酶、蛋白酶、MDH和G6PDH的活性均呈现持续上升的趋势,表明种子代谢活动逐渐增强;而CAT和酸性磷酸酶的活性则呈现先上升后下降的趋势,可能与种子萌发过程中氧化应激和磷酸代谢的动态变化有关。在内源激素方面,GA₃、IAA和BR的含量随层积时间延长呈现上升趋势,表明这些激素在促进种子萌发中起积极作用;CTK的含量则呈现先上升后下降的趋势;而ABA的含量呈现先下降后上升的趋势。此外,激素比值分析显示,IAA/ABA在种子开始萌发后达到峰值,表明生长素与脱落酸的平衡对种子萌发起关键作用;而GA₃/ABA、CTK/ABA和BR/ABA在种子萌发时呈现下降趋势,进一步揭示了激素间动态平衡对种子休眠解除和萌发调控的重要性。（3）本研究从15个样品的转录组测序中共获得92.35Gb Clean Data,通过比较分析,在A＿vs＿C、C＿vs＿E、E＿v s＿G及G＿vs＿H的比较组中,分别鉴定出611（上调369,下调242）、669（上调468,下调201）、1149（上调534,下调615）、2361（上调1108,下调1253）个基因。GO富集显示这些基因主要参与膜的组成部分和DNA结合转录因子活性调控功能。KEGG通路分析揭示,主要与植物激素信号转导和植物-病原体相互作用两条通路密切相关。WGCNA共鉴定出14个模块,其中MEdarkorange2、MEyellow、MEturquoise、MEgreen和MEblue等5个关键模块与种子萌发高度相关,这些模块显著富集于内吞作用、植物激素信号转导等通路。（4）植物激素信号通路解析显示:GA受体（GID）与负调控因子DELLA、AB A受体（PYR/PYL）及下游ABI5/Sn RK2、IAA响应因子（ARF）及早期响应基因（SAU R/IAA/GH3）、CTK双组分调节因子、BAK受体、BR受体（BRI）等关键基因协同参与种子萌发调控网络。此外,还鉴定出2911个转录因子,分别属于210个转录因子家族,其中,C2H2家族数量最多（120个）,其次是AP2/ERF-ERF家族（118个）、RLK-Pelle＿D LSV超家族（108个）和b HLH家族（98个）等。这些发现系统揭示了北沙参种子萌发过程中多激素信号网络的分子互作框架及关键调控节点。结论:综上所述,裸层积处理可以有效打破北沙参种子休眠,并促进种胚的生理变化和胚率提升。在层积过程中,淀粉酶、蛋白酶、MDH和G6PDH活性持续升高,而CAT和酸性磷酸酶活性则呈现先上升后下降的趋势。内源激素分析显示,GA₃、IAA和BR含量随层积时间的延长而增加,CTK含量先上升后下降,ABA含量则先下降后上升。激素比值分析结果表明,IAA/ABA在萌发后达峰值,而GA₃/ABA、CTK/ABA和Br/ABA在萌发时下降。转录组测序共获得92.35Gb Clean Data,鉴定出4086个差异表达基因,主要富集于膜的组成部分、DNA结合转录因子活性、植物-病原体相互作用及植物激素信号转导通路。WGCNA共鉴定出14个模块,其中MEdarkorange2、MEyellow、MEturquoi se、MEgreen和MEblue等5个关键模块与种子萌发高度相关,这些模块显著富集于内吞作用、植物激素信号转导等通路。在植物激素信号转导通路中的关键基因涉及GA受体（G ID）、ABA受体（PYR/PYL）、IAA响应因子（ARF）等。并鉴定出2911个转录因子,C2H2家族数量最多。更多还原

【Abstract】 Objective:Glehniae Radix is the dried root of Coastal glehnia in the family Umbelliferae,which is mainly propagated by seeds,but the seeds have deep dormancy characteristics,which brings a lot of inconvenience to the production.Current research on seed dormancy primarily focuses on dormancy-breaking conditions,while the physiological changes and gene expression patterns remain poorly understood.This study systematically investigates the physiological and biochemical changes during dormancy release in Glehniae Radix seeds and employs transcriptome analysis to identify key genes associated with plant hormone signaling pathways.By elucidating the dynamic expression patterns and functional roles of these genes during dormancy release,we aim to determine how gene expression drives physiological responses to break seed dormancy.These findings will enrich the theoretical framework of seed dormancy regulation and lay a foundation for further molecular mechanism studies.Methods:In this study,we used the seeds of Glehniae Radix as materials,and through dynamic monitoring of the morphological characteristics,enzyme activities and endogenous hormone changes,we elucidated the changing rules of physiological indicators during the process of lifting dormancy of Glehniae Radix seeds,and analyzed the expression of several key genes of plant hormone metabolism and genes related to the lifting of dormancy and germination of seeds by combining with transcriptomic analysis,so as to explain the explanation of the dormancy and germination of Glehniae Radix seeds on the molecular level.We also analyzed the expression of several key genes of plant hormone metabolism and genes related to seed dormancy release and germination in combination with transcriptomics to explain the dormancy and germination of Glehniae Radix seeds at the molecular level.Results:（1）Glehniae Radix seeds were subjected to naked stratification at 5℃.Rapid germination started after 14 days of stratification,and the germination rate reached a peak（100%）at 42 days,while the germination index also reached a value and the average germination time was significantly shortened.The development of seed embryos showed an increasing trend during the lamination process.At the early stage of stratification（0 days）,the seed embryo was small,with an embryo length of 1.0-1.03 mm and an embryo rate of 13.33%;subsequently,the seed embryo grew rapidly from 0 to 42 days,and at 45 days the seed embryo had protruded out of the seed coat,with the embryo length increasing to 4-5 mm,and the embryo rate reaching a maximum value of 39.67%.（2）With the prolongation of stratification time,the activities of amylase,protease,MDH and G6PDH in the seeds of Glehniae Radix showed a continuous rising trend,indicating that the metabolic activities of the seeds were gradually enhanced;whereas the activities of CAT and acid phosphatase showed an increasing and then decreasing trend,which might be related to the dynamic changes of oxidative stress and phosphate metabolism in the process of seed germination.In terms of endogenous hormones,the contents of GA₃,IAA and BR showed an increasing trend with the lamination time,indicating that these hormones played a positive role in promoting seed germination;the contents of CTK showed a first increasing and then decreasing trend;while the contents of ABA showed a first decreasing and then increasing trend.In addition,the hormone ratio analysis showed that IAA/ABA peaked after the seeds started to germinate,indicating that the balance between growth hormone and abscisic acid plays a key role in seed germination;whereas,GA₃/ABA,CTK/ABA and BR/ABA showed a decreasing trend in seed germination,which further revealed the importance of the dynamic balance between hormones in the regulation of seed dormancy lifting and germination.（3）In this study,a total of 92.35 Gb Clean Data was obtained from transcriptome sequencing of 15 samples.By comparative analysis,611（up-regulated 369,down-regulated 242）,669（up-regulated 468,down-regulated 201）,1149（up-regulated 534,down-regulated 615）,2361（up-regulated 1108,down-regulated 1253）genes.GO enrichment revealed that these genes were mainly involved in membrane components and DNA-binding transcription factor activity regulatory functions.KEGG pathway analysis revealed that the two pathways,namely phytohormone signaling and plant-pathogen interactions,were mainly closely related.A total of 14 modules were identified in the WGCNA,of which five key modules,MEdarkorange2,MEyellow,MEturquoise,MEgreen and MEblue,were highly related to seed germination,and these modules were significantly enriched in endocytosis,phytohormone signaling and other pathways.（4）Phytohormone signaling pathway analysis showed that key genes such as GA receptor（GID）and negative regulator DELLA,ABA receptor（PYR/PYL）and downstream ABI5/Sn RK2,IAA response factor（ARF）and early response genes（SAUR/IAA/GH3）,CTK bicomponent regulator,BAK receptor,BR receptor（BRI）and so on.synergistically involved in the seed germination regulatory network.In addition,2,911 transcription factors were identified,belonging to 210transcription factor families,among which the C2H2 family was the most numerous（120）,followed by the AP2/ERF-ERF family（118）,the RLK-Pelle＿DLSV superfamily（108）,and the b HLH family（98）,among others.These findings systematically revealed the molecular interactions framework and key regulatory nodes of the multihormonal signaling network during seed germination in Glehniae Radix.Conclusion:In summary,naked stratification treatment can effectively break the seed dormancy of Glehniae Radix and promote the physiological changes of seed embryo and embryo rate enhancement.During the stratification process,the activities of amylase,protease,MDH and G6PDH continued to increase,while the activities of CAT and acid phosphatase showed a tendency of increasing and then decreasing.The analysis of endogenous hormones showed that GA₃,IAA and BR contents increased with lamination time,CTK contents first increased and then decreased,while ABA contents first decreased and then increased.Hormone ratio analysis showed that IAA/ABA peaked after germination,while GA₃/ABA,CTK/ABA and Br/ABA decreased at germination.A total of 92.35 Gb Clean Data was obtained from transcriptome sequencing,and 4086 differentially expressed genes were identified,which were mainly enriched in membrane components,DNA-binding transcription factor activities,plant-pathogen interactions,and phytohormone signaling pathways.A total of 14 modules were identified in WGCNA,of which MEdarkorange2,MEyellow,MEturquoise,MEgreen and MEblue,and five key modules were highly related to seed germination,which were significantly enriched in endocytosis,phytohormone signaling and other pathways.The key genes in the phytohormone signaling pathway involved GA receptor（GID）,ABA receptor（PYR/PYL）,and IAA response factor（ARF）.And 2911 transcription factors were identified,with the highest number of C2H2family.更多还原