鴉膽子內(nèi)生真菌Br11代謝產(chǎn)物抗TMV活性研究
發(fā)布時(shí)間：2018-10-09 13:34
【摘要】：世界上最早被發(fā)現(xiàn)的病毒就是煙草花葉病毒(Tobacco msaic virus,TMV),TMV嚴(yán)重影響著我國(guó)煙葉的產(chǎn)量和質(zhì)量,每年對(duì)我國(guó)煙草的生產(chǎn)造成巨大的損失,但是到目前為止并沒(méi)有一種可以根治煙草花葉病毒(TMV)的藥劑。前期已有研究者從藥用植物鴉膽子中分離出一種對(duì)煙草花葉病毒(TMV)有很好的抑制作用的化學(xué)物質(zhì)—鴉膽子素D(BruceineD),基于植物內(nèi)生真菌與宿主之間互利共生的關(guān)系,植物內(nèi)生真菌能夠產(chǎn)生與宿主相同或相似的化合物,研究室早期對(duì)鴉膽子植物中的內(nèi)生真菌進(jìn)行了分離純化,并對(duì)這些真菌的代謝產(chǎn)物進(jìn)行抗TMV活性的粗測(cè),本文選取其中一株較好的抗TMV的內(nèi)生真菌Br11進(jìn)行深入的研究。本文對(duì)鴉膽子植物的一株內(nèi)生真菌Br11進(jìn)行代謝產(chǎn)物分離及其抗TMV活性的研究。發(fā)酵該菌株,利用多種色譜分離技術(shù)如硅膠柱層析、RP-C18層析柱、SephadexLH-20葡聚糖凝膠柱層析等對(duì)其發(fā)酵產(chǎn)物進(jìn)行分離純化,得到6種單體化合物和一個(gè)胞外多糖(Extracellular polysaccharide,EPS),分別編號(hào)為 1-7。通過(guò)1H-NMR、13C-NMR波普技術(shù)分析,對(duì)這6種化合物結(jié)構(gòu)進(jìn)行鑒定及查閱文獻(xiàn)對(duì)比,得出化合物1-6分別為DothioreLone A、Dothiorelone B、Dothiorelone C、Dothiorelone H、Cytosporone C、Cytosporone U。對(duì)這些分離出來(lái)的化合物進(jìn)行了抗TMV活性研究,采用半葉枯斑法和葉碟法分別測(cè)定這幾種化合物對(duì)TMV侵染和增殖的抑制活性。結(jié)果表明胞外多糖(組分7)有很強(qiáng)的抗TMV侵染效果,其IC50為0.93μg/mL,但抗TMV增殖效果不明顯�；衔�6 Cytosporone U有較強(qiáng)的抗TMV增殖的作用,其IC50為144.60μg/mL。采用Western-blot和農(nóng)桿菌浸潤(rùn)法進(jìn)一步測(cè)定化合物6在煙草植株內(nèi)抗TMV的生物活性。結(jié)果發(fā)現(xiàn)在濃度為200 μg/mL時(shí),化合物6起到很好的抗TMV-CP表達(dá)、抗TMV增殖和運(yùn)動(dòng)的效果,其有效時(shí)期為7天。本文通過(guò)形態(tài)學(xué)與分子生物學(xué)相結(jié)合的方法對(duì)Br11菌株進(jìn)行了鑒定,該真菌在PDA培養(yǎng)基上菌落生長(zhǎng)迅速,菌絲絲狀長(zhǎng)管,有隔,顏色由白色變?yōu)榈S色再變?yōu)樯铧S色,并產(chǎn)生了黃色的可溶性色素。采用內(nèi)生真菌通用引物ITS 1和ITS 4對(duì)Br11菌株的28S rRNA的保守區(qū)序列進(jìn)行PCR擴(kuò)增并對(duì)擴(kuò)增產(chǎn)物進(jìn)行序列測(cè)定,通過(guò)BLAST分析序列的同源性,發(fā)現(xiàn)該菌株與擬莖點(diǎn)霉屬同源性最高,為99%。通過(guò)構(gòu)建系統(tǒng)發(fā)育樹(shù),發(fā)現(xiàn)Br11菌株與擬莖點(diǎn)霉屬在同一分支上。同時(shí)對(duì)Br11菌株進(jìn)行了不同培養(yǎng)條件下的誘導(dǎo)產(chǎn)孢實(shí)驗(yàn),均沒(méi)有發(fā)現(xiàn)Br11菌孢子的形態(tài)。綜合以上及相關(guān)文獻(xiàn)描述,確定Br11菌株為不產(chǎn)孢的擬莖點(diǎn)霉(Phomopsis sp)真菌。
[Abstract]:The earliest virus found in the world is tobacco mosaic virus (Tobacco msaic virus,TMV), which seriously affects the yield and quality of tobacco in China, and causes huge losses to the production of tobacco in China every year. But so far there is no cure for tobacco mosaic virus (TMV). Brucetin D (BruceineD), a chemical substance that has been isolated from Brucea javanica, a medicinal plant, which has a good inhibitory effect on tobacco mosaic virus (TMV) (TMV), is based on the mutually beneficial symbiotic relationship between endophytic fungi and the host. Plant endophytic fungi can produce the same or similar compounds as the host. The endophytic fungi from Brucea javanica plants were isolated and purified in the early stage, and the metabolites of these fungi were tested for their anti- TMV activity. In this paper, one of the better endophytic fungi Br11 resistant to TMV was selected for further study. In this paper, the metabolites of an endophytic fungus Br11 from Brucea javanica were isolated and its anti TMV activity was studied. The fermentation products of the strain were separated and purified by a variety of chromatographic separation techniques, such as RP-C18 column Sephadex LH-20 gel column chromatography. Six monomers and one extracellular polysaccharide (Extracellular polysaccharide,EPS) were obtained, numbered 1-7, respectively. By 1H-NMR-13C-NMR technique, the structures of the six compounds were identified and compared in literature. The results showed that compounds 1-6 were DothioreLone Dothiorelone (C) Dothiorelone (H) and Cytosporone (C) to Cyporone (U). The anti- TMV activity of these isolated compounds was studied. The inhibitory activities of these compounds on TMV infection and proliferation were determined by semi-leaf blight spot method and leaf disc method respectively. The results showed that the extracellular polysaccharides (component 7) had a strong anti-infection effect of TMV, its IC50 was 0.93 渭 g / mL, but the anti-proliferation effect of TMV was not obvious. Compound 6 Cytosporone U had a strong anti-proliferation effect on TMV, with a IC50 of 144.60 渭 g / mL. The bioactivity of compound 6 against TMV in tobacco plants was further determined by Western-blot and Agrobacterium tumefaciens infiltration method. The results showed that compound 6 had a good effect on the expression of TMV-CP, proliferation and movement of TMV at a concentration of 200 渭 g/mL. The effective period of compound 6 was 7 days. In this paper, the Br11 strain was identified by combining morphology and molecular biology. The fungus grew rapidly on the PDA medium. The mycelium had long tube and septum, and the color changed from white to light yellow and then to dark yellow. Yellow soluble pigment was produced. Endophytic fungi universal primer ITS 1 and ITS 4 were used to amplify the conservative region sequence of 28s rRNA of Br11 strain by PCR and sequencing the amplified products. The homology of BLAST analysis showed that this strain had the highest homology with P. pseudostema, which was 99xuan. By constructing phylogenetic tree, it was found that the Br11 strain was on the same branch as the phyllostachys. At the same time, the spore morphology of Br11 strain was not found in the spore induction experiment under different culture conditions. According to the above and related literatures, Br11 strains were identified as (Phomopsis sp) fungi without sporulation.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
本文編號(hào)：2259523
[Abstract]:The earliest virus found in the world is tobacco mosaic virus (Tobacco msaic virus,TMV), which seriously affects the yield and quality of tobacco in China, and causes huge losses to the production of tobacco in China every year. But so far there is no cure for tobacco mosaic virus (TMV). Brucetin D (BruceineD), a chemical substance that has been isolated from Brucea javanica, a medicinal plant, which has a good inhibitory effect on tobacco mosaic virus (TMV) (TMV), is based on the mutually beneficial symbiotic relationship between endophytic fungi and the host. Plant endophytic fungi can produce the same or similar compounds as the host. The endophytic fungi from Brucea javanica plants were isolated and purified in the early stage, and the metabolites of these fungi were tested for their anti- TMV activity. In this paper, one of the better endophytic fungi Br11 resistant to TMV was selected for further study. In this paper, the metabolites of an endophytic fungus Br11 from Brucea javanica were isolated and its anti TMV activity was studied. The fermentation products of the strain were separated and purified by a variety of chromatographic separation techniques, such as RP-C18 column Sephadex LH-20 gel column chromatography. Six monomers and one extracellular polysaccharide (Extracellular polysaccharide,EPS) were obtained, numbered 1-7, respectively. By 1H-NMR-13C-NMR technique, the structures of the six compounds were identified and compared in literature. The results showed that compounds 1-6 were DothioreLone Dothiorelone (C) Dothiorelone (H) and Cytosporone (C) to Cyporone (U). The anti- TMV activity of these isolated compounds was studied. The inhibitory activities of these compounds on TMV infection and proliferation were determined by semi-leaf blight spot method and leaf disc method respectively. The results showed that the extracellular polysaccharides (component 7) had a strong anti-infection effect of TMV, its IC50 was 0.93 渭 g / mL, but the anti-proliferation effect of TMV was not obvious. Compound 6 Cytosporone U had a strong anti-proliferation effect on TMV, with a IC50 of 144.60 渭 g / mL. The bioactivity of compound 6 against TMV in tobacco plants was further determined by Western-blot and Agrobacterium tumefaciens infiltration method. The results showed that compound 6 had a good effect on the expression of TMV-CP, proliferation and movement of TMV at a concentration of 200 渭 g/mL. The effective period of compound 6 was 7 days. In this paper, the Br11 strain was identified by combining morphology and molecular biology. The fungus grew rapidly on the PDA medium. The mycelium had long tube and septum, and the color changed from white to light yellow and then to dark yellow. Yellow soluble pigment was produced. Endophytic fungi universal primer ITS 1 and ITS 4 were used to amplify the conservative region sequence of 28s rRNA of Br11 strain by PCR and sequencing the amplified products. The homology of BLAST analysis showed that this strain had the highest homology with P. pseudostema, which was 99xuan. By constructing phylogenetic tree, it was found that the Br11 strain was on the same branch as the phyllostachys. At the same time, the spore morphology of Br11 strain was not found in the spore induction experiment under different culture conditions. According to the above and related literatures, Br11 strains were identified as (Phomopsis sp) fungi without sporulation.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S476
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 吳志兵;李向陽(yáng);周翔;楊文清;葉意強(qiáng);宋寶安;楊松;;5-胺基-1-(3-氯-2-吡啶基)-4-(5-甲硫基-1,3,4-口惡二唑-2-基)-吡唑衍生物的合成及抗煙草花葉病毒活性研究[J];中國(guó)科學(xué):化學(xué);2016年11期
2 鄭重;;鴉膽子油對(duì)FOLFOX4方案化療結(jié)直腸癌患者細(xì)胞免疫功能的影響[J];中國(guó)中醫(yī)藥科技;2016年06期
3 程玉鵬;李天聰;林進(jìn)華;陳琦;馬愛(ài)萍;李弘琨;;內(nèi)生真菌與藥用植物宿主間的交流[J];中醫(yī)藥信息;2016年06期
4 姚元勇;張賢;陳仕學(xué);邢明明;舒華;唐幫成;薛偉;;苯并噻唑-β-氨基酸酯類衍生物的合成與抗煙草花葉病毒活性[J];分子科學(xué)學(xué)報(bào);2016年04期
5 田華;陳光勇;董瑜;王靜;王貽鴻;孔凡玉;;毀滅炭疽菌誘抗蛋白的分離純化及對(duì)煙草抗病促生作用[J];中國(guó)煙草科學(xué);2016年04期
6 程玉鵬;李天聰;林進(jìn)華;;產(chǎn)與宿主相同活性成分的內(nèi)生真菌的研究進(jìn)展[J];藥物生物技術(shù);2016年04期
7 初龍;李海燕;;植物內(nèi)生真菌多樣性及其修復(fù)作用研究[J];種子科技;2016年06期
8 牛麗麗;袁肖寒;顧成波;劉紫薇;馬慧;寧文娟;;內(nèi)生真菌產(chǎn)植物次生代謝產(chǎn)物研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2016年11期
9 何海燕;張丹;李斌;;真菌提取物抑制煙草花葉病毒(TMV)研究進(jìn)展[J];應(yīng)用與環(huán)境生物學(xué)報(bào);2016年01期
10 厚凌宇;田兆豐;謝響明;;紫莖澤蘭多糖誘導(dǎo)煙草對(duì)煙草花葉病毒的抗性[J];東北林業(yè)大學(xué)學(xué)報(bào);2015年12期
相關(guān)碩士學(xué)位論文 前1條
1 嚴(yán)凱;新型抗煙草花葉病毒的藥物篩選及作用機(jī)理初步研究[D];貴州大學(xué);2008年
，本文編號(hào)：2259523
本文鏈接：http://sikaile.net/shoufeilunwen/zaizhiyanjiusheng/2259523.html
最近更新
教材專著
