【作者】 刘淼；

【导师】 孙明；

【作者基本信息】 北京林业大学 ， 风景园林学， 2022， 硕士

【摘要】 菊花（Chrysanthemum morifolium）作为中国十大传统名花之一,具有极高的观赏价值和经济价值。目前,土壤盐渍化是全球范围内的生态问题,不仅限制了菊花的生长,还严重阻碍了菊花产业的发展。杂交技术仍然是培育优良抗性品种的主要途径。芙蓉菊（Crossostephium chinense）是分布于我国中南及东南地区的优异耐盐种质资源,但其耐盐机制及育种应用研究较少,甘菊（Chrysanthemum lavandulifolium）对盐胁迫较为敏感,目前对于芙蓉菊的耐盐机制研究较少,对菊花耐盐品种的选育较少。鉴于此,本研究对芙蓉菊、甘菊杂交获得的具有不同耐盐性的杂种后代进行盐胁迫处理,通过测定生理指标和观察生长情况筛选出耐盐差异较大的后代;并对亲本及耐盐差异后代进行生理及解剖学分析、转录组测序,对得到的数据进行富集分析和加权基因共表达网络分析,挖掘芙蓉菊和杂交后代响应盐胁迫的差异表达基因,并对关键候选基因进行表达模式分析,初步解析芙蓉菊和杂交后代的耐盐机制。主要研究结果如下:（1）对芙蓉菊、甘菊及其八个真杂种后代分别用0 mmol·L^-1、500 mmol·L^-1、700mmol·L^-1的Na Cl胁迫处理6h后,测定菊花叶片叶绿素含量、相对电导率、丙二醛含量、脯氨酸含量、抗氧化物酶活、离子含量的变化,确定了适宜的盐胁迫处理浓度为700mmol·L^-1,并对芙蓉菊、甘菊及其后代进行综合评价,筛选出耐盐差异后代CE、CT、CF,同时将其作为后续转录组测序的植物材料;进一步对芙蓉菊、甘菊及其耐盐差异后代CE、CT、CF在700mmol·L^-1Na Cl处理0h、6h、12h、24h,对叶片内的SOD含量、MDA含量、Na⁺和K⁺/Na⁺的变化趋势分析,发现在6h时,SOD含量、MDA含量上升,K⁺/Na⁺下降,故将6h作为测序时间节点;对700mmol·L^-1Na Cl处理后的芙蓉菊、甘菊及其耐盐差异后代的根、茎、叶部位进行解剖学分析,发现根部结构木质部直径增加、皮层厚度增加、细胞体积略有变大,芙蓉菊和后代的韧皮部直径和木质部直径增加,盐胁迫后的海绵组织和栅栏组织受损程度具有差异,叶肉细胞出现吸水膨胀破裂现象,叶片结构变化最为明显,可能与芙蓉菊和后代具有较强耐盐性相关。（2）对盐胁迫下生长的芙蓉菊、甘菊及其耐盐差异后代的叶片进行了转录组测序,共获得168.16G的有效序列,通过De Novo拼接组装,共得到113700条Unigenes,碱基质量值大于30的比例均超过92%。通过GO富集分析发现,GO Term主要与响应盐胁迫信号、氧化还原反应、响应钙离子信号、细胞壁相关物质合成等途径相关。通过KEGG富集分析发现,主要与碳水化合物代谢、植物激素信号转导等代谢途径有关。（3）筛选出芙蓉菊及其杂交后代耐盐的重要调控通路和关键调控基因。以|log2（fold change）|≥2,FDR（false discovery rate）≤0.01为标准进行筛选,共得到15679个差异表达基因,对差异表达基因进行GO富集分析、KEGG富集分析、WGCNA分析。筛选到与碳水化合物代谢、氧化还原及胁迫、细胞壁代谢、植物激素信号转导途径的重要差异表达基因,以及响应非生物胁迫过程的重要转录因子,其中转录因子19个,碳水化合物代谢相关基因25个,植物激素信号转导相关差异基因16个,氧化还原相关及胁迫响应相关的22个,细胞壁代谢相关基因19个。（4）构建芙蓉菊、甘菊及杂交后代响应盐胁迫的调控网络。根据对差异表达基因的分析挑选出关键候选基因进行q RT-PCR表达分析,在相同盐胁迫处理下,关键基因在甘菊、芙蓉菊和后代中的表达模式有明显差异,推测这些基因在甘菊、芙蓉菊和杂交后代对盐胁迫的响应中起着重要作用,q RT-PCR结果也验证了转录组测序结果的可靠性。综上所述,本文研究了盐胁迫下芙蓉菊、甘菊、杂交后代中形态特征、相对电导率、离子含量、抗氧化酶活等生理指标变化,筛选出了具有耐盐差异性的后代;基于转录组测序数据联合加权基因共表达网络分析讨论重要耐盐调控通路,挖掘芙蓉菊、甘菊、杂交后代叶片响应盐胁迫的关键基因,对候选基因进行表达模式差异分析,为今后深入揭示芙蓉菊和杂交后代响应盐胁迫的调控网络奠定理论基础。更多还原

【Abstract】 Chrysanthemum morifolium,as one of the top ten traditional Chinese flowers,has great ornamental and economic value.At present,soil salinization has become a worldwide ecological problem that not only limits the growth of Chrysanthemum morifolium,but also seriously hinders the development of the Chrysanthemum morifolium industry.Hybridisation remains the main way to produce superior resistant varieties.Crossostephium chinense is an excellent salt-tolerant germplasm resource in the genus Chrysanthemum in the broad sense,Chrysanthemum lavandulifolium is more sensitive to salt stress,but there are few studies on the salt tolerance mechanism of Crossostephium chinense and few selections of salt-tolerant varieties of Chrysanthemum morifolium.Because of this,in this study,the progeny of Chrysanthemum lavandulifolium,Crossostephium chinense,and their hybrid offspring with different salt tolerance were treated with salt stress,and the hybrid offspring with greater salt tolerance were screened by measuring physiological indicators and observing growth;Chrysanthemum lavandulifolium,Crossostephium chinense and their salt-tolerant hybrid offspring under the same treatment were subjected to anatomical structure analysis and transcriptome sequencing.The data obtained were subjected to enrichment analysis and weighted gene co-expression network analysis to explore the differentially expressed genes in response to salt stress in Crossostephium chinense and salt-tolerant hybrid offspring,and to analyze the expression patterns of key candidate genes to preliminarily resolve the salt tolerance mechanism of Crossostephium chinense and salt-tolerant hybrid offspring,with a view to enriching the research on the salt tolerance mechanism of plants.We may provide a theoretical basis for the improvement and utilization of salt-tolerant flowers.The main findings of the study are as follows.（1）We treated the Chrysanthemum lavandulifolium,Crossostephium chinense,and their eight true hybrid offspring with 0 mmol·L^-1,500 mmol·L^-1and 700 mmol·L^-1of Na Cl stress for 6h,changes in chlorophyll content,relative conductivity,malondialdehyde content,proline content,superoxide dismutase activity,catalase activity and ion content of leaves were measured separately,and the results showed that under 500 mmol·L^-1 Na Cl treatment,the morphological changes of the plant material were not obvious compared with the control,and the indicators could not accurately distinguish the differences in salt tolerance among the hybrid offspring;under 700 mmol·L^-1 Na Cl treatment,the morphological characteristics showed salt damage,and the trend of changes in the indicators was obvious,we determined the salt stress treatment concentration of 700 mmol·L^-1,and selected the hybrid offspring CE,CT,CF as plant material for subsequent transcriptome sequencing.By analyzing the changes of SOD content,MDA content,Na⁺,and K⁺/Na⁺in CL,CC,CE,CT,and CF after 700 mmol·L^-1Na Cl treatment at 0h,6h,12h,and 24h,6h was selected as the sequencing time point.（2）Transcriptome was performed on the leaves of Chrysanthemum lavandulifolium,Crossostephium chinense,and their hybrid offspring under salt stress,and a total of 168.16G valid sequences were obtained.113,700 unigenes were obtained by De Novo splicing and assembly,with the proportion of base mass values above 30 exceeding 92%.The GO enrichment analysis revealed that the significantly enriched GO Term was mainly associated with redox reaction,metal ion binding,response to calcium ion signal,and response to salt stress.The KEGG enrichment analysis revealed that the significantly enriched KEGG pathways were mainly associated with amino acid metabolism,lipid metabolism,carbohydrate metabolism,plant hormone signal,and other metabolic pathways.（3）Identification of important regulatory pathways and key regulatory genes.We further identified15679 differentially expressed genes using|log2（fold change）|≥2 and FDR（false discovery rate）≤0.01as screening criteria,and performed GO enrichment analysis,KEGG enrichment analysis,and WGCNA analysis on the differentially expressed genes.We screened for differentially expressed genes related to carbohydrate metabolism,oxidation-reduction process and stress,cell wall metabolism,and plant hormone signal pathways,as well as transcription factors important in response to abiotic stress,including19 transcription factors,25 genes related to carbohydrate metabolism,16 genes related to plant hormone signaling,22 genes related to oxidation-reduction process and stress,19 genes related to cell wall metabolism.（4）Constructing regulatory networks of Chrysanthemum lavandulifolium,Crossostephium chinense,and their hybrid offspring in response to salt stress.We selected key candidate genes for q RT-PCR expression pattern analysis based on the analysis of differentially expressed genes,and the expression patterns of the key genes differed in Chrysanthemum lavandulifolium,Crossostephium chinense,and their hybrid offspring under the same salt stress treatment,and we hypothesized that these genes play important roles in the response to salt stress in Crossostephium chinense and their hybrid offspring.The q RT-PCR results also confirmed the reliability of the transcriptome sequencing results.In summary,we investigated the changes in morphological characteristics,relative conductivity,ion content,and antioxidant enzyme activities in Chrysanthemum lavandulifolium,Crossostephium chinense,and their hybrid offspring under salt stress and screened out progeny with differential salt tolerance.The results showed that the expression patterns of the candidate genes were different,which laid a theoretical foundation for future insights into the regulatory networks of Crossostephium chinense and their hybrid offspring in response to salt stress.更多还原