發(fā)布時間：2018-06-24 05:56

  本文選題：瘋草 + 內生真菌��； 參考：《西北農林科技大學》2017年碩士論文

【摘要】：瘋草是豆科棘豆屬和黃芪屬有毒植物的統(tǒng)稱,每年給國內外畜牧業(yè)造成巨大的經(jīng)濟損失。瘋草的主要毒性成分苦馬豆素(swainsonine,SW)是導致采食動物中毒的根本原因。大量研究表明,瘋草中普遍存在著產SW的內生真菌——彎曲牙管蠕孢菌(Undifilum spp),它是瘋草中SW產生的主要原因,瘋草的毒性與瘋草內生真菌Undifilum屬的數(shù)量密切相關。基于本實驗室對我國主要瘋草內生真菌的分離鑒定和遺傳特征研究,對合成SW前體物的篩選,以及對內生真菌的蛋白質組學研究和SW合成相關酶的鑒定,本論文利用無參RNA-Seq技術,主要對SW含量差異較大的瘋草內生真菌(FEL1a-A5和FEL4-F5)進行轉錄組學研究,并利用生物技術平臺篩選出與SW含量相關的差異表達基因,為進一步闡釋瘋草Undifilum屬內生真菌合成SW的機制奠定基礎。主要研究結果如下:1.分別收集20℃培養(yǎng)26 d左右的內生真菌FEL1a-A5和FEL4-F5的菌絲進行RNA-Seq測序,分別得到39M和49M高質量clean reads;從頭組裝(de novo)獲得12,312條Unigenes,將其與四大數(shù)據(jù)庫Nr、Swiss-Prot、KEGG和KOG比對進行基因功能注釋,共有10,393條Unigenes得到注釋;為分析SW含量相關基因提供了參考基因序列。2.差異表達分析共鑒定出差異基因2,663個,其中上調基因有1,651個,下調基因有1,012個。KEGG Pathway顯著性富集分析發(fā)現(xiàn)96條代謝通路,涉及2,224個差異表達基因。GO功能富集分析發(fā)現(xiàn)富集到催化活性和氧化還原活性條目的差異表達基因較多。3.通過相關生物信息學分析,從差異表達基因中鑒定參與棘豆彎曲牙管蠕孢菌(Undifilum oxytropis)中SW生物合成途徑的相關酶,包括酵母氨酸脫氫酶1個、哌可酸氧化酶1個、吡咯啉-5-羧酸還原酶3個、聚酮合酶16個和細胞色素P450 21個。4.從鑒定到的差異基因中挑選11個,進行實時熒光定量PCR(qRT-PCR)驗證。結果表明,1個酵母氨酸脫氫酶基因、1個聚酮合酶基因、1個P450基因表達量下調,1個哌可酸氧化酶基因、3個吡咯啉-5-羧酸還原酶基因、1個聚酮合酶基因、3個P450基因表達量上調,這與轉錄組測序表達趨勢相一致,說明本論文通過RNA-Seq獲得的差異表達基因是可靠的。
[Abstract]:Wild grass is a toxic plant of Echinoides and Astragalus in Leguminosae, which causes huge economic losses to animal husbandry at home and abroad every year. Swainsonine SW, the main toxic component of wild grass, is the root cause of feeding animals poisoning. A large number of studies have shown that there is an endophytic fungus, Undifilum spp), which produces SW, which is the main cause of SW production. The toxicity of wild grass is closely related to the number of the endophytic fungus Undifilum. Based on the studies on the isolation and genetic characteristics of the main endophytic fungi in China, the screening of synthetic SW precursors, the proteomics of endophytic fungi and the identification of the related enzymes of SW biosynthesis in our laboratory. In this paper, we studied the transcriptome of endophytic fungi (FEL1a-A5 and FEL4-F5) with different SW content by using non-ginseng RNA-Seq technique, and screened differentially expressed genes related to SW content by using biotechnology platform. The results provided a basis for further elucidation of the mechanism of SW biosynthesis by endophytic fungi of Undifilum. The main results are as follows: 1. The mycelium of endophytic fungi FEL1a-A5 and FEL4-F5 cultured at 20 鈩，

本文編號：2060322