本文選題：土槿皮乙酸　切入點(diǎn)：芒果炭疽病菌　出處：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文

【摘要】：芒果炭疽病是一種芒果產(chǎn)區(qū)危害普遍且嚴(yán)重的真菌性病害,大量頻繁使用選擇性化學(xué)藥劑防治,易產(chǎn)生抗藥性風(fēng)險(xiǎn)而導(dǎo)致防效降低。土槿皮乙酸(Pseudolaric acid B,PAB)具有良好的農(nóng)用抑菌活性,抑菌譜廣,但其抑菌作用機(jī)制尚不明確,對(duì)其深入研究有望發(fā)現(xiàn)獲得新穎的殺菌作用機(jī)制。本研究以芒果炭疽病菌(Colletotrichum gloeosporioides(Penz.)Sacc.)為研究對(duì)象,以多菌靈(Carbendazim,CBM)為對(duì)照藥劑,采用DAPI染色法和Illumina高通量測(cè)序技術(shù)分別從細(xì)胞水平和基因轉(zhuǎn)錄水平研究PAB對(duì)芒果炭疽病菌有絲分裂的影響,旨在為從分子水平上明確PAB對(duì)芒果炭疽病菌的作用機(jī)制提供線索。主要研究結(jié)果如下:1.核相觀察結(jié)果發(fā)現(xiàn),芒果炭疽病菌在5 mg/L PAB處理下,核分裂時(shí)間被延遲,12h內(nèi)共經(jīng)歷了7次核分裂,比正常萌發(fā)過(guò)程的核分裂少3次,與CBM處理分裂次數(shù)相近。2.芒果炭疽病菌經(jīng)轉(zhuǎn)錄組測(cè)序得到45351798個(gè)clean reads,拼接得到22865個(gè)unigenes,注釋到Nr、Swissprot、KEGG、COG和GO數(shù)據(jù)庫(kù)的Unigene分別是13999個(gè)、79134個(gè)、3931個(gè)、5429個(gè)和2468個(gè),全部的注釋Unigene總計(jì)14100個(gè)。3.基于RPKM法,芒果炭疽病菌在PAB作用下有1783個(gè)基因(8.2%)差異表達(dá),其中726個(gè)基因上調(diào);CBM作用下有1592個(gè)基因(7.4%)差異表達(dá),723個(gè)基因上調(diào);選取25個(gè)基因經(jīng)qRT-PCR驗(yàn)證后的差異表達(dá)趨勢(shì)與測(cè)序結(jié)果基本相符,相關(guān)性R2=0.909。4.差異表達(dá)基因GO和KEEG富集分析表明,PAB和CBM脅迫下,病原菌細(xì)胞組分(cellular component)功能類別內(nèi)顯著富集的前2個(gè)功能條目明顯不同。PAB處理,5個(gè)與代謝和營(yíng)養(yǎng)需求相關(guān)的通路顯著富集(Qvalue0.05),大部分為下調(diào)表達(dá)基因,且未見(jiàn)已知的殺菌劑靶基因;CBM處理后4個(gè)與代謝和核糖體合成相關(guān)代謝通路出現(xiàn)顯著富集。5.PAB脅迫下,多個(gè)與細(xì)胞周期、分裂及細(xì)胞骨架相關(guān)的基因差異表達(dá),包括編碼β1-微管蛋白(TUB1)、細(xì)胞周期調(diào)控蛋白6(Cdc6)、TypeⅡB DNA拓?fù)洚悩?gòu)酶的基因。綜上,PAB與CBM均可減緩芒果炭疽病菌有絲分裂進(jìn)程,但兩者在差異表達(dá)基因及其注釋的生物學(xué)功能和參與的代謝通路有著明顯差異,這表明兩者在分子水平上的作用機(jī)制存在不同之處,推測(cè)PAB抑菌作用可能與其對(duì)微管蛋白與細(xì)胞周期等基因的調(diào)控有關(guān)。
[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，

本文編號(hào)：1673783