本文選題：巫山淫羊藿　切入點：種子休眠　出處：《中國農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：巫山淫羊藿(Epimedium wushanense)為小檗科藥用植物,本論文以巫山淫羊藿種子為試驗材料,研究了種子萌發(fā)及種胚發(fā)育過程對氟啶酮、赤霉素、溫度的響應特征,分析了巫山淫羊藿種子在不同溫度層積條件下脂肪酸代謝和內源激素代謝規(guī)律；結合休眠特性研究和方法篩選,建立了巫山淫羊藿整齊萌發(fā)的休眠解除方法。同時,論文對生理休眠、形態(tài)休眠解除分別涉及到的基因、蛋白質進行了深入研究,探討了巫山淫羊藿種子休眠解除機制。主要研究結果如下：1.巫山淫羊藿種子為非深度復雜型形態(tài)生理休眠種子,變溫層積可解除生理休眠,低溫層積可解除形態(tài)休眠。種子具有內源萌發(fā)抑制物質,系統(tǒng)分離后各組分對黑麥草根的抑制效應表現為丙酮相甲醇相水相乙酸乙酯相,對芽的抑制效應表現為丙酮相乙酸乙酯相正己烷相甲醇相。2.篩選出了最優(yōu)的氟啶酮、赤霉素處理組合：2.5 mg.L-1氟啶酮與400 mg.L-1赤霉素。變溫層積90 d種子,發(fā)芽率、發(fā)芽勢、發(fā)芽指數比對照分別增加33.7%、431.1%、58.1%,平均發(fā)芽時間減少13.6%。變溫層積150 d種子,發(fā)芽率、發(fā)芽勢、發(fā)芽指數分別比對照增加103.3%、1675.0%、25.0%,平均發(fā)芽時間減少19.2%。3.變溫層積可加速巫山淫羊藿種子在后續(xù)低溫層積中的脂肪酸代謝。未經變溫層積和經變溫層積120 d的種子,低溫層積90 d后,脂肪含量分別減少1.9%和2.8%；氟啶酮也可加快脂肪酸代謝速度,3.0mg·L-1、5.0mg·L-1、10 mg·L-1氟啶酮處理變溫120 d的種子,低溫層積90 d后,脂肪含量分別減少3.0%、2.1%和5.4%。4.通過Illumina Hiseq 2500測序平臺,對處于3種不同休眠狀態(tài)的巫山淫羊藿種子進行了RNA-seq測序,共獲得了25.30 Gb可用的測序數據,其中包括79107個非冗余Unigene,平均長度為644.25bp。其中,32384個序列被NR、Swiss-Prot、Pfam、KOG、COG、GO和KEEG七個數據庫注釋到。GO顯著性富集分析與KEGG顯著性富集分析結果表明,生理休眠與形態(tài)休眠解除過程中,差異基因富集到功能類別及代謝途徑具有較高相似性,但基因表達行為不同。5.采用iTRAQ技術,對3種不同休眠狀態(tài)的巫山淫羊藿種子進行蛋白質組學研究,以轉錄組Unigene為蛋白數據庫進行搜庫,共鑒定到550個非冗余蛋白,生理休眠解除過程中共鑒定到369個差異表達蛋白,形態(tài)休眠解除過程中共鑒定到338個差異表達蛋白。6.種子生理休眠解除過程中,磷酸戊糖途徑關鍵酶6-磷酸葡萄糖酸脫氫酶表達水平顯著增加,脂肪酸代謝、線粒體及內質網中相關酶基因表達顯著上調,脂肪酸利用較為活躍。在形態(tài)休眠解除過程中,糖類物質向戊糖轉化能力下降,6-磷酸葡萄糖酸脫氫酶表達顯著下調。脂肪酸代謝途徑中,線粒體和內質網中相關酶基因表達顯著下調,細胞質乙酰CoA羧化酶表達顯著上調,脂肪酸合成活動較為活躍。
[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.
【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S567.239
，

本文編號：1590443