本文關(guān)鍵詞：柑橘褐斑病菌比較基因組和轉(zhuǎn)錄組分析及柑橘綠霉病菌產(chǎn)孢中心調(diào)控途徑和高滲甘油途徑的功能基因研究　出處：《浙江大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 柑橘褐斑病菌 基因組測(cè)序 非必需染色體 線粒體 轉(zhuǎn)錄組 氧化脅迫 柑橘綠霉病菌 產(chǎn)孢中心調(diào)控途徑 PdbrlA PdabaA PdwetA 高滲甘油途徑 Pdos2 甾醇合成

【摘要】：一、柑橘褐斑病菌比較基因組和轉(zhuǎn)錄組分析柑橘褐斑病(Alternaria brown spot, ABS)是部分重要橘,以及這些橘和柚或橘和橙雜交柑橘上的重要病害,引起落葉、落果、枯梢,帶病果實(shí)無法鮮銷,常給感病的柑橘品種生產(chǎn)帶來巨大困難。柑橘褐斑病的病原為交鏈格孢菌橘致病型(A. alternata pathotype tangerine,也稱A. alternata pv. citri),已有的研究發(fā)現(xiàn)該病原菌能夠產(chǎn)生寄主選擇性ACT毒素,而該毒素是病菌致病所必需的；同時(shí)還發(fā)現(xiàn)活性氧解毒系統(tǒng)在病菌致病中也有重要的作用。然而,目前對(duì)柑橘褐斑病菌毒素的合成和調(diào)控過程以及抗氧化調(diào)控網(wǎng)絡(luò)仍缺乏深入的認(rèn)識(shí),對(duì)該病菌的基礎(chǔ)代謝過程,如生長(zhǎng)和繁殖,對(duì)環(huán)境的適應(yīng)以及次生代謝物產(chǎn)生等方面的研究也很少。深入研究柑橘褐斑病菌的生長(zhǎng)、發(fā)育、繁殖、適應(yīng)、次生代謝等基礎(chǔ)生物學(xué)以及致病機(jī)理可為探索全新病害防治途徑,改善現(xiàn)有防治策略提供新的思路,而一個(gè)完整的基因組信息是開展相關(guān)研究的基礎(chǔ)。因此,我們對(duì)一個(gè)來自浙江甌柑的柑橘褐斑病菌菌株Z7進(jìn)行了全基因組測(cè)序,并與已知其他鏈格孢菌的基因組序列進(jìn)行了比較,同時(shí)還分析了H202處理后的轉(zhuǎn)錄組變化,得到以下結(jié)果：1.比較基因組學(xué)研究揭示了柑橘褐斑病菌橘致病型的特異基因柑橘褐斑病菌Z7菌株基因組包含161個(gè)contigs,總長(zhǎng)34.41Mb,平均G+C含量為51.0%。Z7基因組共編碼12062個(gè)基因和115個(gè)tRNA,平均基因長(zhǎng)度1726bp,序列重復(fù)率為0.55%。通過不同致病型交鏈格孢菌株直系同源基因的分類與比較,得到10個(gè)橘致病型特有的基因。這些基因成簇聚集在基因組上,后續(xù)分析認(rèn)為它們是合成ACT毒素的關(guān)鍵組分,決定了柑橘致病型的分化。基因組水平上構(gòu)建的系統(tǒng)進(jìn)化樹與Lawrence等提出的鏈格孢屬下劃分4個(gè)組的觀點(diǎn)相吻合,為鏈格孢屬真菌新分類系統(tǒng)提供了強(qiáng)有力的支撐。交鏈格孢菌橘致病型基因組中發(fā)現(xiàn)了18個(gè)次生代謝基因簇,其中柑橘�；韵嚓P(guān)的ACT毒素基因簇長(zhǎng)91.2kb,包括25個(gè)基因,大部分基因在基因組中都有2-3個(gè)拷貝,推測(cè)在進(jìn)化過程中這些基因發(fā)生了復(fù)制,也暗示Z7具有較強(qiáng)的產(chǎn)生ACT毒素的能力。柑橘褐斑病菌含有較多的細(xì)胞壁降解酶,這與它們死體營(yíng)養(yǎng)的生活方式相一致。2.比較基因組揭示柑橘褐斑病菌非必需染色體起源于鏈格孢菌的祖先我們用比較基因組的方法,獲得了Z7總長(zhǎng)1.88Mb的非必需染色體(Conditionally Dispensable Chromosome, CDC)序列。CDC和EC基因組的組成結(jié)構(gòu)具有明顯的不同,CDC的平均G+C含量是47.7%,序列重復(fù)率為1.23%；Z7CDC編碼525個(gè)蛋白,不含有tRNA。利用GO數(shù)據(jù)庫(kù)分析發(fā)現(xiàn)CDC上的基因主要富集在‘細(xì)胞代謝過程’、‘初級(jí)代謝過程’、‘氧化還原反應(yīng)’和‘大分子代謝過程’等生物學(xué)過程。蛋白家族分析表明CDC上含有13個(gè)碳水化合物酶,29個(gè)分泌蛋白,3個(gè)激酶,21個(gè)轉(zhuǎn)錄因子,25個(gè)轉(zhuǎn)運(yùn)蛋白和13個(gè)細(xì)胞色素P450單加氧酶。密碼子適應(yīng)性指數(shù)(CAI)分析表明EC和CDC基因的密碼子偏好性明顯不同,暗示CDC的進(jìn)化歷史可能與EC不同。通過Ka/Ks分析發(fā)現(xiàn),CDC上的基因都受到強(qiáng)烈的純化選擇。直系同源聚類發(fā)現(xiàn)接近77%的CDC基因的同源基因至少在5個(gè)其他鏈格孢菌種中出現(xiàn),通過比較由24個(gè)鏈格孢菌屬蛋白和29個(gè)其他真菌屬的蛋白組成的兩個(gè)數(shù)據(jù)庫(kù),超過95%的CDC基因都與鏈格孢菌數(shù)據(jù)庫(kù)具有較高的相似性,表明CDC上絕大多數(shù)基因來自于鏈格孢屬真菌的祖先。3.線粒體比較基因組揭示柑橘褐斑病菌與其他種屬線粒體的保守基因在排布上顯示巨大差異柑橘褐斑病菌線粒體基因組大小50,625bp,平均A+T含量為70.8%,含有13個(gè)標(biāo)準(zhǔn)的蛋白基因,2個(gè)核糖體大小亞基和31個(gè)tRNA,線粒體基因組編碼效率為63.7%,整體表現(xiàn)出偏好使用富含A/T的密碼子。腔菌目(Pleosporales)三個(gè)菌株線粒體基因組的基因較為保守,但基因的排布則表現(xiàn)出巨大的差異,暗示它們進(jìn)化歷史不同。用線粒體蛋白構(gòu)建的不同物種系統(tǒng)進(jìn)化樹與核基因組構(gòu)建的進(jìn)化樹-致,說明雖然線粒體基因相對(duì)基因組基因進(jìn)化速度較快,但總體上與物種進(jìn)化趨勢(shì)吻合。不同真菌線粒體內(nèi)基因組內(nèi)含子及基因間區(qū)存在多樣性,是造成線粒體大小差異的重要原因。4.轉(zhuǎn)錄組分析揭示谷胱甘肽系統(tǒng)、過氧化物酶和轉(zhuǎn)運(yùn)蛋白等基因家族在H202脅迫適應(yīng)中發(fā)揮重要作用通過對(duì)柑橘褐斑病菌在H202處理30min后其轉(zhuǎn)錄組表達(dá)變化分析,發(fā)現(xiàn)1108個(gè)上調(diào)表達(dá)基因和498個(gè)下調(diào)表達(dá)基因。差異表達(dá)的基因主要富集在細(xì)胞代謝、氧化還原和細(xì)胞轉(zhuǎn)運(yùn)等生物過程。谷胱甘肽系統(tǒng)、硫氧還蛋白、過氧化氫酶、半胱氨酸過氧化物氧化還原酶等可能是該病菌清除H202的主要成員。此外,我們還發(fā)現(xiàn)轉(zhuǎn)運(yùn)蛋白、激酶家族、轉(zhuǎn)錄因子、細(xì)胞色素P450、泛素和熱激蛋白等在柑橘褐斑病菌H202脅迫適應(yīng)中發(fā)揮重要作用。在非必需染色體上也有29個(gè)基因受到H202的明顯誘導(dǎo),其中包括ACT毒素合成的關(guān)鍵基因聚酮合成酶CDCn|11750。二、柑橘綠霉病菌產(chǎn)孢中心調(diào)控途徑和高滲甘油途徑的功能基因研究柑橘綠霉病(Penicillium digitatum)是柑橘貯藏、運(yùn)輸和銷售環(huán)節(jié)中的主要問題,在我國(guó)每年因綠霉病導(dǎo)致柑橘損失高達(dá)數(shù)百萬噸。迄今,已有3個(gè)柑橘綠霉病菌菌株的全基因組公布,但對(duì)該病菌的生長(zhǎng)發(fā)育、適應(yīng)和致病等的分子機(jī)制了解甚少。形成大量的分生孢子是構(gòu)成病害流行的基礎(chǔ),而抵抗適應(yīng)高滲透勢(shì)等逆境脅迫條件是病菌賴以寄生高糖柑橘果實(shí)的必要基礎(chǔ)。為了解柑橘綠霉病菌產(chǎn)孢和對(duì)滲透勢(shì)的適應(yīng)機(jī)制,本文研究了該病菌的brlA、abA和wetA 3個(gè)產(chǎn)孢中心調(diào)控基因和雙組份組氨酸激酶基因os2的生物學(xué)功能,取得如下結(jié)果：1. PdbrlA、PdabA和PdwetA調(diào)控柑橘綠霉病菌產(chǎn)孢的不同階段病菌繁殖產(chǎn)生的大量無性孢子是綠霉病菌病賴以傳播擴(kuò)散的必要條件。brlA、 abaA和wetA是調(diào)節(jié)分生孢子形成的重要元件。柑橘綠霉病菌中PdbrlA敲除突變體完全喪失了形成分生孢子梗的能力；PdabaA敲除突變體雖能形成分生孢子梗,但所形成的分生孢子�；�,不具有產(chǎn)孢能力；PdwetA的缺失突變體能夠形成正常的分生孢子梗,也可以產(chǎn)孢,但分生孢子的細(xì)胞壁明顯疏松,加厚,分生孢子色素不沉積,分生孢子萌發(fā)延遲,病菌對(duì)滲透脅迫,去垢劑和熱激反應(yīng)的耐受性明顯下降,但對(duì)H202的耐受性卻變強(qiáng)。qRT-PCR結(jié)果表明綠霉菌中存在PdbrlA→PdabA→PdwetA級(jí)聯(lián)調(diào)控模式,而且這種模式可能存在負(fù)反饋調(diào)節(jié)機(jī)制�；虮磉_(dá)譜分析結(jié)果表明：與野生型相比,缺失突變體中的401個(gè)基因下調(diào)表達(dá),144個(gè)基因上調(diào)表達(dá)。Go富集表明下調(diào)表達(dá)的基因功能主要富集在細(xì)胞膜完整性,跨膜轉(zhuǎn)運(yùn)和碳水化合物活性等方面,上調(diào)的基因主要參與細(xì)胞膜轉(zhuǎn)運(yùn)的過程。KEGG分析預(yù)測(cè)PdbrlA與淀粉和蔗糖代謝途徑有關(guān)。根據(jù)煙曲霉和構(gòu)巢曲霉中產(chǎn)孢相關(guān)基因的報(bào)道,我們?cè)诰G霉菌中找到了39個(gè)與產(chǎn)孢相關(guān)的同源基因。其中,12個(gè)基因(abr1、alb1、arp1、arp2、ayg1、aspf4、rodA、rodB、ppoC、axl2、 abaA和wetA)在PdbrlA中表達(dá)明顯下調(diào),2個(gè)基因(flbA和flbB)表達(dá)明顯上升,12個(gè)表達(dá)下調(diào)的基因啟動(dòng)子區(qū)都具有brlA和abaA的轉(zhuǎn)錄結(jié)合位點(diǎn)。2.柑橘綠霉病菌通過Pdos2正調(diào)控甘油含量、負(fù)調(diào)控甾醇含量適應(yīng)高鹽環(huán)境高滲甘油途徑在真核生物體內(nèi)廣泛存在,對(duì)機(jī)體適應(yīng)環(huán)境變化具有極其重要的作用。Os2是該途徑的一個(gè)重要激酶,柑橘綠霉病菌△PdoS2突變體對(duì)NaCl滲透壓和細(xì)胞壁干擾制劑剛果紅和十二烷基苯磺酸鈉的敏感性顯著上升,對(duì)氟咯菌腈和異菌脲兩種殺菌劑的抗性也有部分提高,表明Pdos2參與了高滲透脅迫適應(yīng)、細(xì)胞壁完整性和藥劑抗性等生命過程。然而突變體對(duì)由H202引起的氧化壓的敏感性并沒有明顯改變�！鱌dos2突變體在接種4天后引起的病斑大小相比野生型減少了約25%,表明Pdos2對(duì)維持柑橘綠霉病菌的致病性起到部分作用。0.7M NaCl處理后,野生型菌絲體內(nèi)甘油的含量明顯提高而麥角甾醇的含量卻顯著降低�！鱌doS2突變體菌絲內(nèi)的甘油含量?jī)H有輕微地上升,而麥角甾醇的含量保持與野生型相同的狀態(tài),表明Pdos2參與調(diào)節(jié)的滲透適應(yīng)是與它對(duì)甘油合成的正調(diào)控和麥角甾醇合成的負(fù)調(diào)控作用有關(guān)。Pdos2基因能夠直接或者間接負(fù)調(diào)控甾醇合成途徑關(guān)鍵基因Pderg11C、Pderg1、Pderg3A/B和Pderg25等的表達(dá)。
[Abstract]:A citrus acicola comparative genome and transcriptome analysis of citrus leaf spot (Alternaria brown, spot, ABS) is an important part of orange, orange and grapefruit or orange and orange and citrus hybrid an important disease caused by defoliation, dieback, fruit abscission, fruit can not be sold fresh sick, often susceptible to citrus production bring great difficulties. Citrus brown spot pathogen Alternaria tenuis pathotype (A. alternata pathotype orange tangerine, also known as A. alternata pv. citri), it has been found that the pathogenic bacteria can produce host selectivity of ACT toxin, and the toxin is required for pathogenicity; it was also found that the active oxygen detoxification system also has an important role in pathogenicity. However, the toxin synthesis and citrus acicola regulation and antioxidant regulatory networks is the lack of in-depth understanding of the basic metabolism of the pathogen, such as raw Long and reproduction, and the adaptability of secondary metabolites on the environment are few. The further study of citrus acicola growth, development, reproduction, adaptation, secondary metabolic biology and pathogenic mechanism for exploring new ways to improve the disease prevention and treatment, provide new ideas of prevention strategies, and a complete the genomic information is the basis of related research. Therefore, we performed whole genome sequencing of a Zhejiang mandarin orange citrus acicola strain Z7, and the other known species of Alternaria genomic sequences were compared, and also analyzed the transcriptome changes after H202 treatment, the results are as following: 1. comparison genomics research reveals acicola pathotypes of citrus orange citrus acicola specific gene Z7 strain genome contains 161 contigs, length 34.41Mb, the average content of G+C is 51.0%.Z7 Because group encoding 12062 genes and 115 tRNA genes, the average length of the 1726bp sequence, 0.55%. repetition rate by different pathotype of Alternaria alternata strains of orthologous gene classification and comparison, get the unique 10 orange pathotype genes. These genes are clustered in genome and subsequent analysis and they are considered as the key group synthesis of ACT toxin, determines the differentiation of pathogenicity. Citrus genomic division on the level of phylogenetic tree and Lawrence of Alternaria under 4 groups of views coincide, provides strong support for Alternaria fungi. A new classification system found 18 gene clusters to secondary metabolism Alternaria tangerine pathotype genome, including citrus specific ACT toxin gene cluster length 91.2kb, including 25 genes, most of the genes are 2-3 copies in the genome, which showed that these radicals in the evolutionary process Because the copy also suggests that Z7 has a strong ability to produce ACT toxin. Cell wall degrading enzymes of citrus acicola contain more, with their necrotrophic lifestyle consistent.2. citrus genome reveals acicola nonessential chromosome originated from Alternaria ancestor we use comparative genomics method. The Z7 of 1.88Mb (Conditionally Dispensable Chromosome non essential chromosome, CDC) structure and EC sequence of.CDC genome is obviously different, the average content of G+C is 47.7% CDC, repetition rate of 1.23% Z7CDC encoding sequence; 525 proteins, not containing tRNA. using the GO database analysis found on the CDC gene is mainly concentrated in the "cell metabolism", "primary metabolic process", "redox reaction" and "high molecular metabolism" and other biological processes. Analysis showed that the CDC protein family Containing 13 carbohydrate enzymes, 29 secreted proteins, 3 kinase, 21 transcription factors, 25 transporters and 13 cytochrome P450 monooxygenase. Codon adaptation index (CAI) analysis showed that the codon preference of EC and CDC genes were significantly different, suggesting that the evolutionary history and may CDC EC. The Ka/Ks analysis showed that the CDC genes are subject to strong purifying selection. Orthologous cluster found homologous gene of CDC gene close to 77% in at least 5 other Alternaria species in two databases by comparing 24 Alternaria genus proteins and 29 other fungal genera the protein, more than 95% of the CDC gene with Alternaria database showed high similarity, the vast majority of mitochondrial CDC gene from Alternaria fungi ancestor.3. comparative genomic reveal citrus acicola with other species of mitochondria The conserved genes revealed great differences of citrus acicola mitochondrial genome size 50625bp in the arrangement, the average A+T content was 70.8%, protein gene contains 13 standard, 2 ribosomal subunits and 31 tRNA mitochondrial genome encoding efficiency is 63.7%, the overall performance of a preference for the use of A/T enriched codon. Loculoascomycetes (. Pleosporales) three strains of mitochondrial genes are more conservative, but the gene arrangement showed great differences, suggesting that they are different. The evolutionary history constructed by mitochondrial protein systems of different phylogenetic trees and nuclear genome constructed phylogenetic tree - induced mitochondrial genes, although relative genome evolution speed, but on the whole and the evolutionary trend. There is diversity of different fungal mitochondrial genome intron and intergenic region, is the important reason causing mitochondrial size differences. Transcriptome analysis revealed 4. glutathione peroxidase system and transporter gene family in H202 stress play an important role in the adaptation of citrus acicola in H202 group the expression of 30min after the change analysis, found 1108 up-regulated genes and 498 down regulated genes. Differentially expressed genes mainly enriched in cell metabolism, redox and cellular transport and other biological processes. The glutathione system, thioredoxin, catalase, cysteine peroxiredoxin may be the major members of the bacteria removal of H202. In addition, we also found that the transporter protein kinases, transcription factor, cytochrome P450, ubiquitin and heat shock protein play an important role in stress adaptation in citrus leaf spot of H202. In the non essential chromosome 29 gene was also induced by H202, including ACT toxin synthesis The key gene of polyketide synthase CDCn|11750. two digitatum spore center regulatory pathways and hypertonic glycerol pathways to study functional gene of Citrus green mold (Penicillium digitatum) is the main problem of Citrus storage, transportation and sale, in our country every year because of Citrus green mold caused the loss of up to millions of tons. So far, there 3 strains of Penicillium digitatum genome published, but for the bacteria growth, to understand the molecular mechanism and pathogenic yet. The formation of a large number of conidia are constitute the basis of disease epidemic, and resistance to osmotic potential stress condition is a necessary basis for citrus fruit sugar parasitic bacteria to understand digitatum spore and adaptive mechanism of osmotic potential, was studied in this paper. BrlA, abA and wetA 3 sporulation center regulatory genes and two-component histidine induced The biological function of OS2 gene, the results were as follows: 1. PdbrlA, a large number of asexual spores in different stages of bacteria PdabA and PdwetA regulation digitatum spore breeding is a necessary condition for green mildew disease spreading.BrlA, abaA and wetA are important components of regulating spore formation. Citrus green mold bacteria in PdbrlA knockout mutants completely lost the ability to form conidiophores; PdabaA knockout mutant can form conidiophores, but the formation of conidiophore deformity, does not have the ability to produce spores; deletion mutant PdwetA can form conidiophores are normal, but also can sporulation the cell wall of conidia was loose, thickened, spore pigment deposition, conidia germination delay, resistant to osmotic stress tolerance, detergent and heat shock response decreased significantly, but the tolerance of H202 is The results show that there is strong.QRT-PCR PdbrlA - PdabA - PdwetA cascade control model of green mold, and this pattern may exist negative feedback regulation mechanism. Gene expression spectrum analysis results showed that: compared with the wild type, expression of 401 gene deletion mutants, 144 genes up-regulated expression of.Go gene showed that enrichment downregulated the expression of the main enrichment function in the cell membrane integrity, transmembrane transport and carbohydrate activity, genes involved in cell membrane transport process.KEGG analysis of PdbrlA and starch and sucrose metabolism prediction. According to Aspergillus fumigatus and Aspergillus nidulans sporulation related genes reported, we found 39 and sporulation related homeobox gene in green mold. Among them, 12 genes (abr1, alb1, ARP1, Arp2, ayg1, aspf4, rodA, rodB, ppoC, axl2, abaA and wetA) expression was markedly reduced in PdbrlA, 2 genes (flbA And flbB) was significantly increased in 12, down regulated expression of transcription gene promoter with brlA and abaA binding sites.2. digitatum positive regulation of glycerol content by Pdos2, the negative regulation of sterol content adaptation to high salt environment of hypertonic glycerol pathways in eukaryotic organisms widely exist, has the function of.Os2 is very important an important way to the kinase to the body to adapt to environmental changes, digitatum PdoS2 sensitivity of NaCl mutant on osmotic pressure and cell wall interference preparation of Congo red and twelve sodium increased significantly, resistant to fludioxonil and iprodione two fungicides have increased, indicating that Pdos2 participates in the high adaptation to osmotic stress, cell wall integrity and drug resistance and so on. However the sensitivity of mutant to oxidation caused by H202 pressure did not change significantly. The Pdos2 mutant inoculation in 4 days After the lesion size was reduced by about 25% compared to the wild type, showed that the pathogenicity of Pdos2 to maintain digitatum play a part of.0.7M after NaCl treatment, content of wild type in the mycelium of glycerol increased obviously and ergosterol content was significantly decreased. The content of glycerol PdoS2 mutants in silk only slightly increased. The ergosterol content remains the same as the wild type state that Pdos2 participate in the regulation of the permeability and it is fit for glycerol on.Pdos2 gene negative regulation is regulation of synthesis and synthesis of ergosterol can directly or indirectly negative regulation of sterol biosynthesis pathway genes Pderg11C, Pderg1, expression of Pderg3A/B and Pderg25.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.66
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本文編號(hào)：1355891