【作者】 余永涛；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2009， 博士

【摘要】 疯草是指含有苦马豆素的豆科黄芪属和棘豆属有毒植物,动物疯草中毒病是危害草原畜牧业最严重的问题之一。苦马豆素是疯草的主要毒性成分,它能抑制动物细胞α-甘露糖苷酶和甘露糖苷酶Ⅱ的活性,影响低聚糖的代谢和糖蛋白的加工,导致低聚糖蓄积和糖蛋白合成障碍。中毒动物表现为行为异常、瘫痪、不育、流产、体重下降,严重的发生死亡,造成巨大的经济损失。目前在疯草毒性成分、中毒机理、化学防除和脱毒利用等研究方面取得了一些成绩,但迄今还没有有效控制疯草中毒的方法,致使疯草中毒问题日趋严重,成为草原畜牧业发展的大患。近来的研究表明,美国疯草中的内生真菌能够合成苦马豆素,认为疯草毒素的产生和疯草内生真菌之间存在着必然联系。目前,我国还未见产苦马豆素内生真菌的报道,我国疯草中是否也存在参与疯草毒素合成的内生真菌还需进一步证实。疯草内生真菌的研究,将为我国动物疯草中毒研究开辟新的领域,为动物疯草中毒病的防治和疯草的利用提供新的科学依据。本文主要对中国疯草内生真菌的分离与鉴定、产苦马豆素内生真菌的筛选、ITS-5.8S rDNA序列及其遗传进化、遗传多态性、固体培养特性等方面进行研究。1.疯草内生真菌的分离与初步鉴定对中国的10种黄芪属、棘豆属植物进行了内生真菌分离和初步鉴定,共分离到42株真菌,主要为埃里格孢属、链格孢属、未知种属真菌3类。其中从变异黄芪、小花棘豆、毛瓣棘豆、冰川棘豆、甘肃棘豆、黄花棘豆、茎直黄芪等7种疯草中分离到11株埃里格孢属真菌;通过扫描电镜观察,进一步确定该11株埃里格孢属真菌为疯草内生真菌。此外还分离到14株链格孢属真菌和17株未知真菌。本研究为产苦马豆素内生真菌的筛选奠定了基础。2.产苦马豆素内生真菌的筛选对苦马豆素提取和分离方法进行比较,筛选出适于微量样品分析的苦马豆素提取和分离方法。利用该方法对植物样品和真菌菌丝进行处理,通过薄层色谱法、气相色谱法、气相色谱-质谱联用法,从42株真菌中筛选出11株含有苦马豆素的真菌,该类真菌均为埃里格孢属内生真菌,分别来自变异黄芪、小花棘豆、毛瓣棘豆、冰川棘豆、甘肃棘豆、黄花棘豆、茎直黄芪等7种疯草,其菌丝中苦马豆素的含量为10.83±1.31～571.11±7.79 mg/kg;在发酵培养试验中,进一步证实该类真菌能够在人工发酵培养的条件下产生苦马豆素,其发酵液中的苦马豆素含量为2～85 mg/L,具有液体发酵生产苦马豆素的潜能。本研究首次证实中国疯草中存在能够产生苦马豆素的内生真菌,将为从控制产苦马豆素内生真菌生长角度防治动物疯草中毒病提供依据。3.产苦马豆素内生真菌的ITS-5.8S rDNA序列测定及其遗传进化分析应用PCR法对11株分离自中国7种重要疯草的产苦马豆素内生真菌的ITS-5.8SrDNA序列进行了扩增,并测定了核苷酸序列,确定该类真菌的ITS-5.8S rDNA片段长度为600～602 bp,不同菌株间仅相差1～3个核苷酸位点,遗传进化分析表明该类真菌与Embellisia oxytropis OIB9、Embellisia oxytropis OsL12、Embellisia oxytropis DAOM237697、Embellisia sp.OY2.21等真菌遗传距离最近,根据形态学、遗传学分析结果和文献报道,确定该11株真菌均为棘豆埃里格孢菌(Embellisia oxytropis)。本研究对我国7种疯草的产苦马豆素内生真菌进行了种级划分和鉴定,为我国疯草内生真菌的研究奠定了真菌分类基础,为进一步深入研究疯草内生真菌的遗传多态性提供了依据。4.产苦马豆素内生真菌的遗传多态性研究应用RAPD和IGS-RFLP技术对11株产苦马豆素真菌的遗传多态性进行了研究。通过对20个随机引物的筛选,发现仅有OPA-1、OPA-18、OPA-20等3个引物扩增出的不同真菌的条带数量和长度出现了较小的差异,说明该11株真菌间的亲缘关系非常密切,通过本研究中使用的引物无法将其区分。不同菌株的IGS序列均具有DraⅠ、HindⅢ、SmaⅠ等酶切位点,且数量相同,但IGS序列的长度及其限制性酶切片段的长度发生了较大的变异,表现为内部的酶切位点位置的不同,可以确定该11株真菌间存在着种间差异,可能分别为棘豆埃里格孢菌的不同亚种。通过IGS-RFLP技术,能够实现对不同疯草内生真菌分离株的明显区分,为产苦马豆素疯草内生真菌遗传变异规律的研究提供了依据。5.产苦马豆素内生真菌的固体培养特性研究对从黄花棘豆、茎直黄芪中分离的产苦马豆素内生真菌F5、G3的固体培养特性进行了初步研究,筛选出适宜F5、G3生长的最适碳源、最适氮源、最适pH分别为燕麦片、蛋白胨、pH 4.4。当以无机氮为氮源,最适碳氮比为100:1,当以有机氮为氮源,最适碳氮比为200:1。本试验为产苦马豆素内生真菌的液体发酵工艺研究提供依据,为进一步研究疯草内生真菌合成苦马豆素的途径、合成机理奠定了基础。更多还原

【Abstract】 Locoweeds are toxic plants of the genera Astragalus and Oxytropis containing swainsonine. Locoism is one of the most widespread poisonous plant problems. Swainsonine is the principal agent responsible for locoism in animals and an potent inhibitor of lysosomeα-mannosidase and Golgi mannosidase II in cell. Inhibition of the mannosidases in cells of mammals leads to an accumulation of mannose-rich oligosaccharides in the lysosomes or interferes with the synthesis of complex glycoproteins in cells. Affected animals appear as behavioural abnormality, paralysis, infertility, abortion, weight loss and eventually death. Consumption of locoweed by animals results tremendous economic losses. Up to now, the toxicology, pathology of locoism have been detailed described, but it is remain a great problem to protect animals from this poisonous disease, locoism has become the most important disease on the vast rangeland. Recently, the fungal endophytes are isolated frequently from locoweeds in the US and showed to produce swainsonine in vitro. There is a strong relationship between the presence of endophytes and the swainsonine concentration of their host plants. The endophytes are believed to be responsible at least partially for the toxicity of locoweeds. However, it is not documented that swainsonine-producing fungal endophyte infecting locoweeds in China. The research about swainsonine-producing fungal endophyte will offer a new reference for controlling and using locoweed. This paper was main about the isolation and identification of fungal endophytes from locoweeds in China, determination of swainsonine-producing fungal endophytes, phylogenetic relationships based on ITS-5.8S rDNA sequences, genetic polymorphism and cultural characteristics on solid media of fungal endophytes.1. Isolation and identification of fungal endophytes from locoweedsTen species of the genera Astragalus and Oxytropis were conducted for isolation of fungal endophytes. Forty-two fungal isolates were obtained and classified three groups, such as Alternaria, Embellisia, and undetermined fungi. Eleven Embellisia fungi were isolated respectively from seven species of locoweeds that including Astragalus variabilis, Oxystropis glabra, Oxytropis sericopetala, Oxytropis glacialis, Oxytropis kansuensis, Oxytropis ochrocepala and Astragalus strictus. The Embellisia fungi were determined as fungal endophytes of these plants by scanning electron microscope. Moreover, fourteen Alternaria fungi and seventeen undetermined fungi were also harvested in this assay. The study was important for the further determination of swainsonine-producing fungal endophytes in these locoweeds.2. Determination of swainsonine-producing fungal endophytes from locoweedsThe methods of extraction and isolation about swainsonine were evaluate, the optimal method were determined and used to the treatment of plant and fungi samples. Eleven of 42 isolates were confirmed containing swainsonine by TLC, GC and GC-MS. The fungi containing swainsonine were Embellisia spp, isolated from Astragalus variabilis, Oxystropis glabra, Oxytropis sericopetala, Oxytropis glacialis, Oxytropis kansuensis, Oxytropis ochrocepala and Astragalus strictus, respectively. The swainsonine contents of the fungal mycelia were 10.83±1.31 mg/kg to 571.11±7.79 mg/kg. Subsequently, the fungal endophytes were also verified producing swainsonine in liquid fermentation in vitro. The concentrations of swainsonine in fermentation liquors of endophytes were 2～85 mg/L. The endophytes is promising to be used to producing swainsonine. It is the first document about swainsonine-producing fungal endophytes from locoweeds in China. The study offers a new possible avenue for dealing with locoism by controlling the endophytes instead of locoweed plants.3. Sequencing of ITS-5.8S rDNA and phylogenetic relationships based on ITS-5.8S rDNA sequences of swainsonine-producing fungal endophytesThe amplification and sequencing were conducted for the ITS-5.8S rDNA regions of eleven swainsonine-producing fungal endophytes. The sizes of amplified fragments were 600 to 602 bp. The minute differences, variations of one to three nucleotide, occurred among the ITS-5.8S rDNA sequences of eleven fungal endophytes. Phylogenetic analysis based on ITS-5.8S rDNA sequences showed the fungal endophytes were closely related to Embellisia oxytropis OIB9、Embellisia oxytropis OsL12、Embellisia oxytropis DAOM 237697、Embellisia sp. OY2.21. Eleven swainsonine-producing fungal endophytes were identified as Embellisia oxytropis according to the morphology, genetics of endophytes and associated documents. The taxa of the fungal endophytes were determined on the level of species. This study offers the basis for the further research of genetic polymorphism of swainsonine-producing fungal endophytes.4. Genetic polymorphisms of swainsonine-producing fungal endophytesThe analysis about genetic polymorphisms of swainsonine-producing endophytic fungi were performed by RAPD and IGS-RFLP. Twenty random primers were screened in this assay. It was found that a smaller difference occurred in the RAPD spectrum of different isolates only using primer OPA-1, OPA-18 and OPA-20. The results indicated that eleven swainsonine-produing fungal endophytes had a very close genetic relationship and could not be distinguished by the primers used in this study. IGS regions of all isolates had Dra I, Hind III, Sma I restriction sites and the number of restriction sites were same. However, the sizes of IGS sequences and restriction fragments from different fungi were various which exhibited the different locations of internal restriction sites. This means that there were differences among the 11 fungal species, which could be different subspecies of Embellisia oxytropis. IGS-RFLP was a potent method for distinguishing different isolates of swainsonine-producing fungal endophytes. This study provided the basis for the study on genetic variation of swainsonine-producing fungal endophytes from locoweeds.5. Characteristics of growth of swainsonine-producing fungal endophytes on solid mediaA preliminary study were performed about the Characteristics of growth of swainsonine-producing fungal endophytes (F5, G3) isolated from Oxytropis ochrocepala and Astragalus strictus on solid media. The optimal carbon source, nitrogen source, pH were oatmeal, peptone pH 4.4. The optimal ratio of carbon and nitrogen was 100:1, 200:1 respectively when inorganic nitrogen or organic nitrogen as nitrogen source. This study provides a basis for the study on the fermentation and the synthetic mechanism of swainsonine of endophytic fungi.更多还原