基于转录组测序探究土槿皮乙酸对芒果炭疽病菌有丝分裂的影响

发布时间：2018-03-27 23:34

  本文选题：土槿皮乙酸　切入点：芒果炭疽病菌　出处：《黑龙江八一农垦大学》2017年硕士论文

【摘要】：芒果炭疽病是一种芒果产区危害普遍且严重的真菌性病害,大量频繁使用选择性化学药剂防治,易产生抗药性风险而导致防效降低。土槿皮乙酸(Pseudolaric acid B,PAB)具有良好的农用抑菌活性,抑菌谱广,但其抑菌作用机制尚不明确,对其深入研究有望发现获得新颖的杀菌作用机制。本研究以芒果炭疽病菌(Colletotrichum gloeosporioides(Penz.)Sacc.)为研究对象,以多菌灵(Carbendazim,CBM)为对照药剂,采用DAPI染色法和Illumina高通量测序技术分别从细胞水平和基因转录水平研究PAB对芒果炭疽病菌有丝分裂的影响,旨在为从分子水平上明确PAB对芒果炭疽病菌的作用机制提供线索。主要研究结果如下:1.核相观察结果发现,芒果炭疽病菌在5 mg/L PAB处理下,核分裂时间被延迟,12h内共经历了7次核分裂,比正常萌发过程的核分裂少3次,与CBM处理分裂次数相近。2.芒果炭疽病菌经转录组测序得到45351798个clean reads,拼接得到22865个unigenes,注释到Nr、Swissprot、KEGG、COG和GO数据库的Unigene分别是13999个、79134个、3931个、5429个和2468个,全部的注释Unigene总计14100个。3.基于RPKM法,芒果炭疽病菌在PAB作用下有1783个基因(8.2%)差异表达,其中726个基因上调;CBM作用下有1592个基因(7.4%)差异表达,723个基因上调;选取25个基因经qRT-PCR验证后的差异表达趋势与测序结果基本相符,相关性R2=0.909。4.差异表达基因GO和KEEG富集分析表明,PAB和CBM胁迫下,病原菌细胞组分(cellular component)功能类别内显著富集的前2个功能条目明显不同。PAB处理,5个与代谢和营养需求相关的通路显著富集(Qvalue0.05),大部分为下调表达基因,且未见已知的杀菌剂靶基因;CBM处理后4个与代谢和核糖体合成相关代谢通路出现显著富集。5.PAB胁迫下,多个与细胞周期、分裂及细胞骨架相关的基因差异表达,包括编码β1-微管蛋白(TUB1)、细胞周期调控蛋白6(Cdc6)、TypeⅡB DNA拓扑异构酶的基因。综上,PAB与CBM均可减缓芒果炭疽病菌有丝分裂进程,但两者在差异表达基因及其注释的生物学功能和参与的代谢通路有着明显差异,这表明两者在分子水平上的作用机制存在不同之处,推测PAB抑菌作用可能与其对微管蛋白与细胞周期等基因的调控有关。
[Abstract]:Mango anthracnose is a common and serious fungal disease in mango producing area. It is easy to produce the risk of drug resistance and lead to the decrease of the control effect. Pseudolaric acid acid (PAB) has good agricultural bacteriostatic activity and wide spectrum of antimicrobial activity, but the mechanism of its bacteriostatic action is not clear. In this study, Colletotrichum gloeosporioidesPenz. Sacc. was used as the research object, and Carbendaziman CBM was used as control. The effects of PAB on mitosis of Mango anthracnose were studied at cell level and gene transcription level by DAPI staining and Illumina high throughput sequencing, respectively. The main results were as follows: 1. Nuclear phase observation showed that Mango anthracnose was treated with 5 mg/L PAB. The mitotic time was delayed for 12 hours, which was 3 times less than that of normal germinating. The number of cleavage of mango anthracnose was similar to that of CBM. 45351798 clean were obtained by transcriptome sequencing, 22865 unigeneses were obtained by splicing, and the Unigene of Nrrus SwissprotKEGGG COG and go database were 13999,79134, 3931, 5429 and 2468 respectively. The total number of annotated Unigene was 14100. 3. Based on the RPKM method, there were 1783 genes of anthracnose treated with PAB. Among them, 726 genes upregulated 1592 genes under the action of PAB, and 1592 genes were up-regulated, and 723 genes were up-regulated. The trend of differential expression of 25 genes verified by qRT-PCR was basically consistent with the sequencing results, and the correlation between R2O0.909.4. The enrichment analysis of differentially expressed genes go and KEEG showed that the two genes were stressed by PAB and CBM. The first two functional items that were significantly enriched in the functional category of cellular component of pathogenic bacteria were significantly different from the treatment of .PAB. Five pathways related to metabolism and nutritional requirements were significantly enriched in Qvalue0.05, most of which were down-regulated expression genes. Moreover, four metabolic pathways associated with metabolism and ribosomal biosynthesis were significantly enriched after CBM treatment with no known bactericide target gene. 5. Under PAB stress, multiple differentially expressed genes related to cell cycle, division and cytoskeleton were found. It includes the genes encoding 尾 1-tubulin TUB1, cell cycle regulatory protein 6c6, type 鈪，

本文编号：1673783