基于RNA-Seq技术的Undifilum oxytropis中苦马豆素含量相关基因的分析

发布时间：2018-06-24 05:56

  本文选题：疯草 + 内生真菌　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：疯草是豆科棘豆属和黄芪属有毒植物的统称,每年给国内外畜牧业造成巨大的经济损失。疯草的主要毒性成分苦马豆素(swainsonine,SW)是导致采食动物中毒的根本原因。大量研究表明,疯草中普遍存在着产SW的内生真菌——弯曲牙管蠕孢菌(Undifilum spp),它是疯草中SW产生的主要原因,疯草的毒性与疯草内生真菌Undifilum属的数量密切相关。基于本实验室对我国主要疯草内生真菌的分离鉴定和遗传特征研究,对合成SW前体物的筛选,以及对内生真菌的蛋白质组学研究和SW合成相关酶的鉴定,本论文利用无参RNA-Seq技术,主要对SW含量差异较大的疯草内生真菌(FEL1a-A5和FEL4-F5)进行转录组学研究,并利用生物技术平台筛选出与SW含量相关的差异表达基因,为进一步阐释疯草Undifilum属内生真菌合成SW的机制奠定基础。主要研究结果如下:1.分别收集20℃培养26 d左右的内生真菌FEL1a-A5和FEL4-F5的菌丝进行RNA-Seq测序,分别得到39M和49M高质量clean reads;从头组装(de novo)获得12,312条Unigenes,将其与四大数据库Nr、Swiss-Prot、KEGG和KOG比对进行基因功能注释,共有10,393条Unigenes得到注释;为分析SW含量相关基因提供了参考基因序列。2.差异表达分析共鉴定出差异基因2,663个,其中上调基因有1,651个,下调基因有1,012个。KEGG Pathway显著性富集分析发现96条代谢通路,涉及2,224个差异表达基因。GO功能富集分析发现富集到催化活性和氧化还原活性条目的差异表达基因较多。3.通过相关生物信息学分析,从差异表达基因中鉴定参与棘豆弯曲牙管蠕孢菌(Undifilum oxytropis)中SW生物合成途径的相关酶,包括酵母氨酸脱氢酶1个、哌可酸氧化酶1个、吡咯啉-5-羧酸还原酶3个、聚酮合酶16个和细胞色素P450 21个。4.从鉴定到的差异基因中挑选11个,进行实时荧光定量PCR(qRT-PCR)验证。结果表明,1个酵母氨酸脱氢酶基因、1个聚酮合酶基因、1个P450基因表达量下调,1个哌可酸氧化酶基因、3个吡咯啉-5-羧酸还原酶基因、1个聚酮合酶基因、3个P450基因表达量上调,这与转录组测序表达趋势相一致,说明本论文通过RNA-Seq获得的差异表达基因是可靠的。
[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，

本文编号：2060322