【作者】 唐明娟；

【导师】 郭顺星； 林忠平；

【作者基本信息】 中国协和医科大学 ， 生药学， 2004， 博士

【摘要】 一、菌根真菌在金线莲栽培中应用及其共生机理研究 福建金线莲Anoectochillus roxburghii(AR)和台湾金线莲A．formosanus(AF)同属兰科开唇兰属濒危药用植物。在自然条件下兰科植物常与真菌形成互惠互利的共生关系。据此，将菌根真菌AR—15和AR—18接种到无菌状态下生长在蛭石基质上的福建金线莲组培苗根部，发现接菌处理对其鲜重的增加有极显著促进作用(P＜0.01)，提高69.3％(AR-15)和74％(AR-18)；对其苗高也有极显著影响(P＜0.01)，分别提高33.99％和31.1％。因此在福建金线莲和台湾金线莲的人工栽培中，利用菌根真菌AR-18实现了两种金线莲的人工栽培。 为了进一步说明菌根真菌对两种金线莲生长的影响，我们将两种金线莲种入花盆中。结果显示福建金线莲接种18号菌根真菌后鲜重和干重比对照分别提高45.3％和21.7％。台湾金线莲分别提高40.4％和26.2％。同时测定了两种金线莲果糖、蔗糖、多糖和可溶性总糖的含量。结果表明这四种干物质的含量较不接菌的对照组相比均不同程度地得以提高。十一种无机元素中铬和铁的吸收增加，对其它九种元素的影响随金线莲种类和栽培基质不同而变化。 为从生化角度了解菌根真菌促进两种金线莲生长的原因，我们测定了几丁质内切酶酶活、几丁质外切酶酶活、β-1，3-葡聚糖酶酶活、多酚氧化酶酶活和苯丙氨酸解氨酶的酶活。结果栽培在草炭土基质中的台湾金线莲接菌处理后酶活为对照的1.13倍-1.29倍；而福建金线莲酶活为对照的1.03倍-1.52倍。在蛭石基质上，菌根真菌对四种酶活的影响比草炭土大。如对台湾金线莲来说，这四种酶活为对照的1.27倍．1.72倍。对福建金线莲来说也是蛭石基质上的影响大，四种酶活为对照的1.17倍-1.84倍。所以在不同基质中这四种酶的酶活在AR—18号真菌处理的两种金线莲中均不同程度地被提高，而这四种酶本身与提高植物抗性有关。 为了寻找菌根真菌促进金线莲生长的分子机理，采用CTAB法从富含多糖和酚类物质的福建金线莲中提取出高质量的RNA作为模板，利用mRNA差异显示技术的优化方法，如银染变性聚丙烯酰胺凝胶，地高辛标记反向Northern的两个cDNA探针，优化PCR扩增程序等，分离到差异表达的七个阳性片段。从基因表达水平来说，五个在接菌处理后表达水平上调，一个表达受抑制，一个为菌刺激特异表达。测序后进行同源性比较，两个为己知基因，五个为未知基因。更多还原

【Abstract】 一、 Studies on the application of mycorrhizal fungui in the culture of Anoectochillus and their symbiotic mechanismAnoectochillus roxburghii and A. formosanus, belonging to Orchidaceae, are rare medicinal plants. Nearly every species of plants in this family is known to form a mycorrhizal relationship with fungi. Based on the characteristic, we cultured the seedlings of A. roxburghii with mycorrhizal fungi AR-15 and AR-18 respectively in the vermiculite medium. The effects of AR-15 and AR-18 on promoting the growth of the two medicinal plants were very significant. Fresh weight of A. roxburghii inoculated with fungi AR-15 and AR-18 were increased by 69.3% and 74% (P<0.01)respectively compared with the non-inoculated ones. And the effects of fungi AR-15 and AR-18 on height were very significant (P<0.01). The height of A. roxburghii inoculated with fungi AR-15 and AR-18 were increased by 33.99% and 31.1% respectively. The artificial cultures of A. roxburghii and A. formosanus with mycorrhizal fungi AR-18 were successfully fulfilled in our laboratory.In order to further demonstrate the application effects of mycorrhizal fungi in the growth of the two medicinal plants, we planted them with strain AR-18 in peat and vermiculite of flowerpots in greenhouse. The results showed that fresh weight and dry weight of A. roxburghii inoculated with strain AR-18 were increased by 45.3% and by 21.7% respectively compared with the non-inoculated ones, and A. formosanus were by 40.4% and by 26.2%respectively. The contents of some sugar compounds in the two plants were also measured. The contents of fructose、 saccharose、 polysaccharide and soluble sugar could be increased in seedlings of the two plants when they were planted with strain AR-18. The contents of Cr and Fe also were increased in seedlings cultured with AR-18, while the effects of mycorrhizal fungus on other inorganic elements in the two plants were different along with different species of Anoectochillus and planted medium.Activities of endochitinase, ectochitinase, β-1,3-glucase, PPO and PAL in A. roxburghii and A. formosanus were measured for understanding the function of mycorrhizal fungi in accelerating the growth of the two medicinal plants, . In peat medium, the activities of four enzymes in A. formosanus were1.13- 1.29 times compared with the controls, and in A. roxburghii the ratios were 1.03-1.52 times respectively. In vermiculite medium, the effect of strain AR-18 on four enzymes was larger than that in peat medium. For example, the activities of four enzymes in A.更多还原