【作者】 陈娜；

【导师】 李春杰；

【作者基本信息】 兰州大学 ， 草业科学， 2011， 博士

【摘要】 内生真菌与多种植物都存在共生的现象并且能够显著提高宿主植物生物胁迫以及非生物胁迫的抵抗能力。已有研究证明内生真菌能够增强宿主植物低温胁迫的耐受性,然而关于内生真菌提高宿主植物低温抗性分子机制方面研究尚无报道。本研究以醉马草(Achnatherum inebrians)-内生真菌(Neotyphodium gansuense)共生体为研究对象,主要研究内生真菌对醉马草低温萌发的影响以及通过表达谱差异分析和蛋白组学研究揭示共生体在分子水平发生的适应性变化。研究结果表明,在15℃-25℃,带有内生真菌(E+)、不带内生真菌(E-)醉马草种子萌发率无明显差异(P>0.05),然而在10℃条件下,E+、E-醉马草种子萌发率显著降低,而E+醉马草种子萌发率(43%)明显高于E-醉马草种子(8%)。进一步利用Solexa测序方法建立了10℃条件下E+E-醉马草种子萌发基因差异表达谱,发现在E+、E-样本中有152个基因发生了显著性差异表达(|log2Ratio|≥2, P<0.001),以E-样本为对照,在E+样本中有109个基因表达量增加,有43个基因表达量明显下降。利用RACE方法克隆了6个差异表达基因的3’末端cDNA序列并且采用RT-PCR方法进行了这些基因的差异表达分析,结果与Solexa测序结果完全一致。对这些差异农达基因的进一步功能聚类分析表明它们参与了多种代谢过程,其中主要包括：生物碱的合成,脂肪酸代谢,蛋白周转,胁迫相应基因,核酸代谢,转妹以及信号转导,未和功能基因以及参与其他多种代谢途径的基因。其中值得关注的是有关麦角碱生物合成的前体色氨酸合成的基因大量表达,比如磷酸烯醇式丙酮酸羧化酶、脱氢硫胺素合酶、磷酸莽草酸羧基转移酶以及转酮醇酶等。另外脂肪酸代谢途径相关基因在E+醉马草种子低温萌发过程中大量表达。膜脂中不饱和脂肪酸在高等植物低温抵抗过程中起着至为关键的作用,在E+醉马草中有10个参与脂肪酸代谢途径的基因发生了差异表达,其中包括参与了不饱和脂肪酸的合成两个酰基去饱合酶同源基因表达量显著增加。总的来说,内生真菌显著提高其宿主醉马草种子低温条件下的萌发能力。内生真菌侵染导致参与多个代谢途径的一系列基因发生了差异表达,推测这些基因协同作用,进而提高了醉马草的低温抵抗能力。进一步利用双向电泳进行内生真菌导致醉马草低温耐受能力的差异蛋白组学研究,发现10个差异表达蛋白,以E-样本为对照,在E+样本中其中8个蛋白的表达量显著提高,生物信息学分析表明这些蛋白是主要参与的能量代谢的蛋白以及胁迫诱导应激蛋白。这些关键基因和蛋白的挖掘,对于我们深入理解禾草-内生真菌共生体相互作用关系以及在分子水平揭示内生真菌提高宿主植物抗逆境胁迫能力的作用机理奠定了良好的基础。更多还原

【Abstract】 Plants have developed mutualistic symbiosis with diverse fungal endophytes increasing fitness by conferring abiotic and biotic stress tolerance. However, the molecular regulation mechanisms involved in stress resistance remain largely unknown. Drunken horse grass (Achnatherum inebrians), an important perennial bunchgrass in China, is naturally occurring symbiosis with an asexual fungus Neotyphodium gansuense. The effect of temperature on seed germination was determined for drunken horse grass infected (E+) vs. non-infected (E-) by N. gansuense. Our results indicated that E+ seeds showed a higher germination rate under low temperature (10℃) condition compared with E- seeds, whereas under relatively higher temperature (15,20 and 25℃) conditions, no obviously difference were detected between the E+ and E- seeds. To gain insight into the molecular mechanisms involved in the low temperature resistance of E+ grass. Solexa deep-sequencing and proteomics were used to identify candidate genes and proteins showing differential expressed level. Firstly.152 differentially expressed tags with a log2 ratio>2 or<-2 (P<0.001) were identified, representing 109 up-regulated and 43 down-regulated genes, which were classified into 8 functional categories. Many genes were found to be association with low temperature response, such as genes participating in biosynthesis of alkaloids and unsaturated fatty acids etc. In turn, RT-PCR analysis showed that the expression patterns of six genes are agreement with the Solexa analysis. Secondly, about 10 differently expressed proteins were sueeessfully identified in our research. Those proteins mainly involved in the energy metabolism and stress response. In conclusion, this study provides comprehensive gene expression changes induced by Neotyphodium endophyte infection response to low temperature which are essential for understanding the molecular basis of endophyte-enhanced plant improvements.更多还原