巫山淫羊藿种子休眠机制及解除方法研究

发布时间：2018-03-09 22:03

  本文选题：巫山淫羊藿　切入点：种子休眠　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：巫山淫羊藿(Epimedium wushanense)为小檗科药用植物,本论文以巫山淫羊藿种子为试验材料,研究了种子萌发及种胚发育过程对氟啶酮、赤霉素、温度的响应特征,分析了巫山淫羊藿种子在不同温度层积条件下脂肪酸代谢和内源激素代谢规律；结合休眠特性研究和方法筛选,建立了巫山淫羊藿整齐萌发的休眠解除方法。同时,论文对生理休眠、形态休眠解除分别涉及到的基因、蛋白质进行了深入研究,探讨了巫山淫羊藿种子休眠解除机制。主要研究结果如下：1.巫山淫羊藿种子为非深度复杂型形态生理休眠种子,变温层积可解除生理休眠,低温层积可解除形态休眠。种子具有内源萌发抑制物质,系统分离后各组分对黑麦草根的抑制效应表现为丙酮相甲醇相水相乙酸乙酯相,对芽的抑制效应表现为丙酮相乙酸乙酯相正己烷相甲醇相。2.筛选出了最优的氟啶酮、赤霉素处理组合：2.5 mg.L-1氟啶酮与400 mg.L-1赤霉素。变温层积90 d种子,发芽率、发芽势、发芽指数比对照分别增加33.7%、431.1%、58.1%,平均发芽时间减少13.6%。变温层积150 d种子,发芽率、发芽势、发芽指数分别比对照增加103.3%、1675.0%、25.0%,平均发芽时间减少19.2%。3.变温层积可加速巫山淫羊藿种子在后续低温层积中的脂肪酸代谢。未经变温层积和经变温层积120 d的种子,低温层积90 d后,脂肪含量分别减少1.9%和2.8%；氟啶酮也可加快脂肪酸代谢速度,3.0mg·L-1、5.0mg·L-1、10 mg·L-1氟啶酮处理变温120 d的种子,低温层积90 d后,脂肪含量分别减少3.0%、2.1%和5.4%。4.通过Illumina Hiseq 2500测序平台,对处于3种不同休眠状态的巫山淫羊藿种子进行了RNA-seq测序,共获得了25.30 Gb可用的测序数据,其中包括79107个非冗余Unigene,平均长度为644.25bp。其中,32384个序列被NR、Swiss-Prot、Pfam、KOG、COG、GO和KEEG七个数据库注释到。GO显著性富集分析与KEGG显著性富集分析结果表明,生理休眠与形态休眠解除过程中,差异基因富集到功能类别及代谢途径具有较高相似性,但基因表达行为不同。5.采用iTRAQ技术,对3种不同休眠状态的巫山淫羊藿种子进行蛋白质组学研究,以转录组Unigene为蛋白数据库进行搜库,共鉴定到550个非冗余蛋白,生理休眠解除过程中共鉴定到369个差异表达蛋白,形态休眠解除过程中共鉴定到338个差异表达蛋白。6.种子生理休眠解除过程中,磷酸戊糖途径关键酶6-磷酸葡萄糖酸脱氢酶表达水平显著增加,脂肪酸代谢、线粒体及内质网中相关酶基因表达显著上调,脂肪酸利用较为活跃。在形态休眠解除过程中,糖类物质向戊糖转化能力下降,6-磷酸葡萄糖酸脱氢酶表达显著下调。脂肪酸代谢途径中,线粒体和内质网中相关酶基因表达显著下调,细胞质乙酰CoA羧化酶表达显著上调,脂肪酸合成活动较为活跃。
[Abstract]:Epimedium wushanense, a medicinal plant of Berberidae, was used in this paper to study the response of seed germination and embryo development to flurazone, gibberellin and temperature. The metabolism of fatty acids and endogenous hormones of Epimedium epimedii seeds at different temperatures was analyzed, and the dormancy release method of Epimedium epimedii in Wushan was established according to the study of dormancy characteristics and the screening of methods. In this paper, the genes and proteins involved in physiological dormancy and morphological dormancy release were studied. The mechanism of dormancy release of Epimedium epimedii seeds in Wushan was discussed. The main results were as follows: 1. The seeds of Epimedium in Wushan were non-deep complex morphological physiological dormancy seeds, and the physiological dormancy could be relieved by changing the temperature layer. Low temperature stratification can relieve morphological dormancy. Seeds have endogenous germination inhibitor. The inhibition effect of each component on ryegrass root after systematic separation is acetone phase methanol phase water phase ethyl acetate phase. The inhibitory effect on buds was as follows: acetone phase, ethyl acetate phase, n-hexane phase methanol phase .2.The optimal fluazidone was screened out. The combination of gibberellin treatment and #number0# mg.L-1 gibberellin was used. Compared with the control, the germination index increased 33.7% and 431.1%, and the average germination time decreased 13.60.The seed germination rate, germination potential and germination rate were 150 days. The germination index increased by 103.3% than that of the control, and the average germination time decreased by 19.22.3.The fatty acid metabolism of Herba Epimedii seeds in Wushan was accelerated during the subsequent low-temperature stratification. The seeds without or after 120 days of caloric stratification were stratified at low temperature for 90 days. The fat content was decreased by 1.9% and 2.8, and the fatty acid metabolism rate was accelerated by 3.0mg 路L ~ (-1) ~ 5.0mg 路L ~ (-1) F _ (-1) and 10 mg 路L ~ (-1) flurididone. After 90 days of low temperature stratification, the fat content was decreased by 3.0mg 路L ~ (-1) and 5.4mg 路L ~ (-1), respectively. By Illumina Hiseq 2500 sequencing platform, the fat content was decreased by 3.0mg 路L ~ (-1) and 5.4mg 路L ~ (-1), respectively. The seeds of Herba Epimedii in three different dormant states were sequenced by RNA-seq, and the available sequencing data of 25.30 GB were obtained. Among them, 79107 non-redundant Unigenees, with an average length of 644.25bp.Of them, 32384 sequences were annotated by seven databases of NRX Swiss-ProtPfamkokov Coggo and KEEG. The results of significant enrichment analysis and KEGG significant enrichment analysis showed that physiological dormancy and morphological dormancy were in the process of dormancy and morphological dormancy release. Different genes were enriched to functional classes and metabolic pathways, but gene expression behavior was different. ITRAQ technique was used to study the proteomics of three kinds of dormant seeds of Epimedium Epimedii. A total of 550 non-redundant proteins were identified using transcriptional Unigene as protein database, and 369 differentially expressed proteins were identified during physiological dormancy release. A total of 338 differentially expressed proteins were identified during the course of morphological dormancy release. During the course of seed physiological dormancy release, the expression of 6-phosphogluconate dehydrogenase, a key enzyme of the pentose phosphate pathway, was significantly increased and fatty acid metabolism was observed. In mitochondria and endoplasmic reticulum, the expression of related enzyme genes was up-regulated, and fatty acid utilization was more active in mitochondria and endoplasmic reticulum. The expression of gluconate dehydrogenase (GDH) was significantly down-regulated in mitochondria and endoplasmic reticulum (ER), and the expression of acetyl CoA carboxylase in cytoplasm was up-regulated in mitochondria and endoplasmic reticulum (ER). Fatty acid synthesis is more active.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.239
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本文编号：1590443