【作者】 陈颖；

【导师】 文李；

【作者基本信息】 长沙理工大学 ， 工程硕士（专业学位）， 2014， 硕士

【摘要】 油菜(Brassica napus L.)是重要的经济作物,我国是世界第一大油菜生产国。菌核病是我国油菜生产的第一大病害,造成重大经济损失。芥子油苷为十字花科植物中重要的次生代谢产物之一,能被植物内源黑芥子酶水解,生成多种生物活性产物,可增强植物的抗病性。本文以抗病油菜湘油15及其感病近等基因系XYNL为实验材料,分析油菜芳香族芥子油苷的抗菌核病作用,并找出其抗菌核病关键基因。为验证两品种油菜对菌核病的抗性差异,本文分别采用二氨基联苯胺(Diaminobenzidine, DAB)染色法、氯化硝基四氮唑蓝(Nitrotetrazolium Blue chloride,NBT)染色法和台盼蓝染色法观察两品种油菜接种菌核菌后叶片H2O2、O2-、死细胞积累情况,采用考马斯亮蓝染色法观察菌核菌菌丝侵入情况。结果表明,接种相同时间内,湘油15叶片内活性氧、死细胞和菌丝积累量均大于XYNL,说明湘油15对菌核菌的抗性比XYNL强,湘油15为抗病品种,近等基因系XYNL为感病品种。芥子油苷水解产物中异硫氰酸酯为主要抗病功能物质,而苯乙基芥子油苷是油菜中唯一的芳香族芥子油苷,因此,为验证油菜芳香族芥子油苷对抗菌核病的作用,本文运用化学合成的苯乙基异硫氰酸酯(2-Phenylethyl isothiocyanate, PEITC),对菌核菌进行体外抑制实验,并通过气相色谱法分析感、抗病品种油菜内源PEITC含量。结果显示,化学合成PEITC能显著抑制菌核菌菌丝的体外生长,且PEITC浓度越大,对菌核菌菌丝生长的抑制作用越显著,当PEITC浓度为14μg/mLL时,抑制率达到100%；接种72h后,抗病油菜叶片中的PEITC含量由接种前的0.125μg/g升高至0202μg/g,增幅为61.6%；而感病油菜叶片中的PEITC含量由接种前的0.192 μg/g升高至0.204μg/g,其增幅仅为6.3%；说明PEITC与油菜抗菌核病有关,油菜芳香族芥子油苷具有抗菌核病作用。为寻找油菜芳香族芥子油苷合成及水解通路中与抗菌核病相关的关键基因,以接种菌核菌后,感、抗病油菜叶片为研究对象,采用荧光定量PCR技术分析该通路相关酶基因的表达量变化。接种菌核菌后,除黑芥子酶(Myrosinase, MYR)基因外,感、抗病油菜叶片中所有酶基因表达量均显著增加；其中,抗病油菜叶片中CYP83B1 (cytochrome P450, family 83, subfamily B, polypeptide 1) 和ST5a (Sulfotransferase)表达量的最大增长倍数均显著高于感病油菜,分别比感病油菜增加了219%和375%；MYR基因表达量减小,且呈先减小后增大的变化趋势,其原因可能为MYR与芥子油苷反应生成的水解产物对MYR的表达具反馈调节作用,推测MYR可能是该通路的限速酶,MYR基因在该通路中起重要作用：综合以上结果可知,CYP83B 1、ST5a和MY R基因是油菜芳香族芥子油苷合成及水解通路中与抗菌核病相关的关键基因。本研究表明,油菜芳香族芥子油苷具有抗菌核病作用,在其合成及水解通路中,CYP83B1、ST5a和MYR基因与抗菌核病密切相关。该研究结果将为调控相关基因在油菜中的表达提供理论基础,为菌核病的生物防治提供新思路,并为抗病油菜的育种提供理论基础。更多还原

【Abstract】 Brassica napus L., or canola, is an important economic crop, and it is widely planted in China. Sclerotinia sclerotiorum causes the most serious diseases in B. napus L., and leads to significant economic losses. Glucosinolate, a kind of plant secondary metabolite, distributes in Cruciferous, Capparaceae and Papaya, and it can be hydrolyzed by endogenous myrosinase, producing a series of products with biological activities. These hydrolysis products can improve the plants’ resistance to diseases. In this paper, leaves from a resistant line Xiangyou 15 and a susceptible near-isogenic lines XYNL were chosen to analyzed the resistant effects of the aromatic glucosinolate in the B. napus L. against S. sclerotiorum. The key genes involved in disease defence were found and further analyses.were adopted to confirm their resistaant function.To compare the resistance of the two varieties, volume of H2O2, O2-, dead cells, and the invaded mycelium in the leaves of the two varieties of canola were detected by utilizing DAB, NBT, Trypan blue, and Coomassie blue stainings respectively.Isothiocyanates are the main functional substance in the hydrolysis products of glucosinolates.Phenethyl glucosinolate is the only one aromatic glucosinolate in B. napus L. leaves. To verify the effect of rapeseed aromatic glucosinolates on S. sclerotiorum, we investigated the inhibition of chemical synthetic 2-phenethyl isothiocyanate (PEITC) on S. sclerotiorum in vitro, and analysed endogenous PEITC content of two varieties by gas chromatography. The results showed that the chemical synthetic PEITC could significantly inhibit mycelial growth of S. sclerotiorum in vitro. The inhibition of mycelial growth of S. sclerotiorum increased with of PEITC concentration. When the concentration of PEITC was higher than 14μg/mL, the inhibition rate on S. sclerotiorum reached 100%; Endogenous PEITC concentration in the resistant line leaves increased from 0.125μg/g to 0.202μg/g, which had an amplification of 61.6% at 72h after inoculation. However, endogenous PEITC concentration in the susceptible line leaves only rose from 0.192μg/g to 0.204μg/g, which just increased by 6.3% at the same time point. Thus this indicated that PEITC was related to the resistance of canola against S. sclerotiorum. We supposed that the aromatic glucosinolate in B. napus L. might affect on the growth of S. sclerotiorum.In order to find the key genes related to resistance, the expression level changes of enzymes in the aromatic glucosinolate synthesis and hydrolysis pathway after inculation were analysed by using real-time quantitative PCR. The results showed that gene expression levels of all the enzymes in the pathway had a significant increase in canola leaves after inoculation except for Myrosinase (MYR). Gene expression levels of CYP83B1 (cytochrome P450, family 83, subfamily B, polypeptide 1) and ST5a (Sulfotransferase) in resistant line leaves showed more significant increse than that in the susceptible one, rising by 219%and 375% respectively compared to the susceptible canola. MYR gene had a reduction in gene expression level, and showed a tendency to increase after the first decrease, which probably owing to the feedback regulation of hydrolysis products of glucosinolates. This inclined that myrosinase might be the key regulation enzyme in the given pathway and the MYR gene had an important role in the pathway. Our results indicated that CYP83B1 gene, ST5a gene and MYR gene might be the key genes in aromatic glucosinolate synthesis and hydrolysis pathway and associated with the defence against S. sclerotiorum.The results in this study showed that the aromatic glucosinolate of B. napus L. played an important role on resistance against S. sclerotiorum in B. napus L. It was also verified that some important genes involved in the aromatic glucosinolate synthesis and hydrolysis pathway, i. e. CYP83B1 gene, ST5a gene and MYR gene, contributed to defence against S. sclerotiorum. Therefore, our research results might provide a new idea for the biological control of S. sclerotiorum and laid a theoretical fundation for the breeding of resistant canola. It could also improve the production of B. napus L.and increase economic benefits by regulating the expression of these genes.更多还原