本文關(guān)鍵詞： 匍匐翦股穎 立枯絲核菌 轉(zhuǎn)錄組 差異表達(dá)基因 抗病機(jī)制　出處：《中國農(nóng)業(yè)科學(xué)》2017年17期 　論文類型：期刊論文

【摘要】：【目的】確定匍匐翦股穎(Agrostis stolonifera)接種立枯絲核菌(Rhizoctonia solani)后基因種類和表達(dá)量在轉(zhuǎn)錄水平的變化規(guī)律,明確草坪草病原菌侵染響應(yīng)的關(guān)鍵基因�！痉椒ā抠橘媵骞煞f生長14 d后采用麥粒培養(yǎng)物接種立枯絲核菌,接種3 d后選取感病葉片和未接種葉片提取RNA,進(jìn)行轉(zhuǎn)錄組高通量測序,然后利用生物信息學(xué)分析,用Trinity組裝匍匐翦股穎轉(zhuǎn)錄組,以組裝子為參考,以|log2(fold change)|1,q-value0.005為閾值選取感病和健康匍匐翦股穎葉片轉(zhuǎn)錄組的差異表達(dá)基因,并用i TAK軟件分析其轉(zhuǎn)錄因子家族及表達(dá)變化,與植物R基因庫進(jìn)行blast分析R蛋白分類、利用Mapman軟件分析生物脅迫信號通路相關(guān)基因的表達(dá)變化�！窘Y(jié)果】高通量測序得到125 253 092條高質(zhì)量待分析reads。經(jīng)Trinity從頭組裝后,得到466 761條轉(zhuǎn)錄本。過半數(shù)的轉(zhuǎn)錄本長度為700 bp以上,組裝結(jié)果 N50=1 100 bp。使用CD-HIT選擇334 212條轉(zhuǎn)錄本(所有轉(zhuǎn)錄本的71.60%)作為Unigene,平均長度573 bp,N50=791 bp。接種后植物比接種前植物基因有7 937個(gè)上調(diào)表達(dá),1 570個(gè)下調(diào)表達(dá)。上調(diào)基因中296個(gè),下調(diào)基因有142個(gè)都可被定義為轉(zhuǎn)錄因子,分布在58個(gè)轉(zhuǎn)錄因子家族中,其中鋅指蛋白包含轉(zhuǎn)錄因子C2H2最多,有54個(gè),C3H次之,為22個(gè)。差異基因中451個(gè)可定義為植物R蛋白表達(dá)基因,可分為33類,其中包含NBS-LRR結(jié)構(gòu)域的抗病蛋白、LRR受體蛋白激酶、ABC-2類型的轉(zhuǎn)運(yùn)蛋白、U-box結(jié)構(gòu)域蛋白激酶和熱激蛋白這5類基因變化最顯著。差異基因中大量上調(diào)表達(dá)基因可富集在病原識別、活性氧消除、信號傳導(dǎo)、細(xì)胞凋亡、病程相關(guān)蛋白等生物脅迫相關(guān)基因類別,下調(diào)基因顯著富集在植物生長發(fā)育相關(guān)類別和通路。q RT-PCR驗(yàn)證了隨機(jī)挑選差異基因的表達(dá)量變化,均與RNA-seq分析結(jié)果一致,其中包括12個(gè)C2H2轉(zhuǎn)錄因子基因,10個(gè)C3H轉(zhuǎn)錄因子基因,以及12個(gè)R蛋白基因。【結(jié)論】病原菌侵染后,引起匍匐翦股穎大量基因表達(dá)變化,其中轉(zhuǎn)錄因子、R蛋白以及抗性相關(guān)基因多為上調(diào)表達(dá),作用為抑制病原菌擴(kuò)散,而生長發(fā)育相關(guān)基因表達(dá)下調(diào),這些進(jìn)程共同使匍匐翦股穎產(chǎn)生對立枯絲核菌的先天基礎(chǔ)抗性。
[Abstract]:[objective] to determine the variation of gene types and expression levels at transcription level after inoculation of Agrostis stolonifera with Rhizoctonia solaniae (Rhizoctonia solani). [methods] after 14 days of growth of creeping bentgrass, Rhizoctonia solani was inoculated with wheat grain culture, and RNA was extracted from susceptible and uninoculated leaves 3 days later, and high throughput sequencing was carried out. Then using bioinformatics analysis, Trinity was used to assemble the transcriptome of bentgrass, with the assembler as reference, and log2(fold exchange) 1nq-value0.005 as the threshold to select the differentially expressed genes of leaf transcriptome of susceptible and healthy creeping bentgrass. I TAK software was used to analyze the transcription factor family and its expression changes, and blast was used to analyze the classification of R protein with the plant R gene bank. Mapman software was used to analyze the expression changes of genes associated with biological stress signaling pathway. [results] 125,253,092 high-quality READswere obtained by high-throughput sequencing. 466,761 transcripts were obtained. More than half of the transcripts were more than 700 BP in length. CD-HIT was used to select 334,212 transcripts (71.60 of all transcripts) as Unigenes.The average length of CD-HIT was 573bpN ~ (50,791) bp.There were 7,937 up-regulated genes and 1,570 down-regulated genes after inoculation, and 296 of them were up-regulated genes. 142 down-regulated genes can be defined as transcription factors, distributed in 58 transcription factor families, among which zinc finger protein contains the transcription factor C2H2, followed by 54 C3H. There are 451 genes that can be defined as plant R protein expression genes, which can be classified into 33 classes. The most significant changes were found in the five types of genes including NBS-LRR domain resistance protein kinase, ABC-2 type transporter, U-box domain protein kinase and heat shock protein. A large number of up-regulated genes in differentially expressed genes can be enriched in pathogen recognition. Reactive oxygen species (Ros) elimination, signal transduction, apoptosis, pathogenesis-related proteins, and down-regulated genes were significantly enriched in plant growth and development related classes and pathways. Q RT-PCR confirmed the changes in the expression levels of randomly selected differentially expressed genes. All the results were consistent with the results of RNA-seq analysis, including 12 C2H2 transcription factor genes, 10 C3H transcription factor genes and 12 R protein genes. [conclusion] after infection by pathogenic bacteria, a large number of genes expressed in creeping bentgrass were changed. The transcription factor R protein and resistance-related genes were upregulated and inhibited the spread of pathogen, but the expression of growth-development related genes was down-regulated. These processes led to the innate basic resistance against Rhizoctonia solani in creeping bentgrass.
【作者單位】： 甘肅農(nóng)業(yè)大學(xué)草業(yè)學(xué)院/草業(yè)生態(tài)系統(tǒng)教育部重點(diǎn)實(shí)驗(yàn)室/中-美草地畜牧業(yè)可持續(xù)發(fā)展研究中心;
【基金】：國家自然科學(xué)基金(31360583)
【分類號】：S436.8

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