柑橘褐斑病菌比较基因组和转录组分析及柑橘绿霉病菌产孢中心调控途径和高渗甘油途径的功能基因研究

发布时间：2017-12-30 18:17

  本文关键词：柑橘褐斑病菌比较基因组和转录组分析及柑橘绿霉病菌产孢中心调控途径和高渗甘油途径的功能基因研究　出处：《浙江大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 柑橘褐斑病菌 基因组测序 非必需染色体 线粒体 转录组 氧化胁迫 柑橘绿霉病菌 产孢中心调控途径 PdbrlA PdabaA PdwetA 高渗甘油途径 Pdos2 甾醇合成

【摘要】：一、柑橘褐斑病菌比较基因组和转录组分析柑橘褐斑病(Alternaria brown spot, ABS)是部分重要橘,以及这些橘和柚或橘和橙杂交柑橘上的重要病害,引起落叶、落果、枯梢,带病果实无法鲜销,常给感病的柑橘品种生产带来巨大困难。柑橘褐斑病的病原为交链格孢菌橘致病型(A. alternata pathotype tangerine,也称A. alternata pv. citri),已有的研究发现该病原菌能够产生寄主选择性ACT毒素,而该毒素是病菌致病所必需的；同时还发现活性氧解毒系统在病菌致病中也有重要的作用。然而,目前对柑橘褐斑病菌毒素的合成和调控过程以及抗氧化调控网络仍缺乏深入的认识,对该病菌的基础代谢过程,如生长和繁殖,对环境的适应以及次生代谢物产生等方面的研究也很少。深入研究柑橘褐斑病菌的生长、发育、繁殖、适应、次生代谢等基础生物学以及致病机理可为探索全新病害防治途径,改善现有防治策略提供新的思路,而一个完整的基因组信息是开展相关研究的基础。因此,我们对一个来自浙江瓯柑的柑橘褐斑病菌菌株Z7进行了全基因组测序,并与已知其他链格孢菌的基因组序列进行了比较,同时还分析了H202处理后的转录组变化,得到以下结果：1.比较基因组学研究揭示了柑橘褐斑病菌橘致病型的特异基因柑橘褐斑病菌Z7菌株基因组包含161个contigs,总长34.41Mb,平均G+C含量为51.0%。Z7基因组共编码12062个基因和115个tRNA,平均基因长度1726bp,序列重复率为0.55%。通过不同致病型交链格孢菌株直系同源基因的分类与比较,得到10个橘致病型特有的基因。这些基因成簇聚集在基因组上,后续分析认为它们是合成ACT毒素的关键组分,决定了柑橘致病型的分化。基因组水平上构建的系统进化树与Lawrence等提出的链格孢属下划分4个组的观点相吻合,为链格孢属真菌新分类系统提供了强有力的支撑。交链格孢菌橘致病型基因组中发现了18个次生代谢基因簇,其中柑橘专化性相关的ACT毒素基因簇长91.2kb,包括25个基因,大部分基因在基因组中都有2-3个拷贝,推测在进化过程中这些基因发生了复制,也暗示Z7具有较强的产生ACT毒素的能力。柑橘褐斑病菌含有较多的细胞壁降解酶,这与它们死体营养的生活方式相一致。2.比较基因组揭示柑橘褐斑病菌非必需染色体起源于链格孢菌的祖先我们用比较基因组的方法,获得了Z7总长1.88Mb的非必需染色体(Conditionally Dispensable Chromosome, CDC)序列。CDC和EC基因组的组成结构具有明显的不同,CDC的平均G+C含量是47.7%,序列重复率为1.23%；Z7CDC编码525个蛋白,不含有tRNA。利用GO数据库分析发现CDC上的基因主要富集在‘细胞代谢过程’、‘初级代谢过程’、‘氧化还原反应’和‘大分子代谢过程’等生物学过程。蛋白家族分析表明CDC上含有13个碳水化合物酶,29个分泌蛋白,3个激酶,21个转录因子,25个转运蛋白和13个细胞色素P450单加氧酶。密码子适应性指数(CAI)分析表明EC和CDC基因的密码子偏好性明显不同,暗示CDC的进化历史可能与EC不同。通过Ka/Ks分析发现,CDC上的基因都受到强烈的纯化选择。直系同源聚类发现接近77%的CDC基因的同源基因至少在5个其他链格孢菌种中出现,通过比较由24个链格孢菌属蛋白和29个其他真菌属的蛋白组成的两个数据库,超过95%的CDC基因都与链格孢菌数据库具有较高的相似性,表明CDC上绝大多数基因来自于链格孢属真菌的祖先。3.线粒体比较基因组揭示柑橘褐斑病菌与其他种属线粒体的保守基因在排布上显示巨大差异柑橘褐斑病菌线粒体基因组大小50,625bp,平均A+T含量为70.8%,含有13个标准的蛋白基因,2个核糖体大小亚基和31个tRNA,线粒体基因组编码效率为63.7%,整体表现出偏好使用富含A/T的密码子。腔菌目(Pleosporales)三个菌株线粒体基因组的基因较为保守,但基因的排布则表现出巨大的差异,暗示它们进化历史不同。用线粒体蛋白构建的不同物种系统进化树与核基因组构建的进化树-致,说明虽然线粒体基因相对基因组基因进化速度较快,但总体上与物种进化趋势吻合。不同真菌线粒体内基因组内含子及基因间区存在多样性,是造成线粒体大小差异的重要原因。4.转录组分析揭示谷胱甘肽系统、过氧化物酶和转运蛋白等基因家族在H202胁迫适应中发挥重要作用通过对柑橘褐斑病菌在H202处理30min后其转录组表达变化分析,发现1108个上调表达基因和498个下调表达基因。差异表达的基因主要富集在细胞代谢、氧化还原和细胞转运等生物过程。谷胱甘肽系统、硫氧还蛋白、过氧化氢酶、半胱氨酸过氧化物氧化还原酶等可能是该病菌清除H202的主要成员。此外,我们还发现转运蛋白、激酶家族、转录因子、细胞色素P450、泛素和热激蛋白等在柑橘褐斑病菌H202胁迫适应中发挥重要作用。在非必需染色体上也有29个基因受到H202的明显诱导,其中包括ACT毒素合成的关键基因聚酮合成酶CDCn|11750。二、柑橘绿霉病菌产孢中心调控途径和高渗甘油途径的功能基因研究柑橘绿霉病(Penicillium digitatum)是柑橘贮藏、运输和销售环节中的主要问题,在我国每年因绿霉病导致柑橘损失高达数百万吨。迄今,已有3个柑橘绿霉病菌菌株的全基因组公布,但对该病菌的生长发育、适应和致病等的分子机制了解甚少。形成大量的分生孢子是构成病害流行的基础,而抵抗适应高渗透势等逆境胁迫条件是病菌赖以寄生高糖柑橘果实的必要基础。为了解柑橘绿霉病菌产孢和对渗透势的适应机制,本文研究了该病菌的brlA、abA和wetA 3个产孢中心调控基因和双组份组氨酸激酶基因os2的生物学功能,取得如下结果：1. PdbrlA、PdabA和PdwetA调控柑橘绿霉病菌产孢的不同阶段病菌繁殖产生的大量无性孢子是绿霉病菌病赖以传播扩散的必要条件。brlA、 abaA和wetA是调节分生孢子形成的重要元件。柑橘绿霉病菌中PdbrlA敲除突变体完全丧失了形成分生孢子梗的能力；PdabaA敲除突变体虽能形成分生孢子梗,但所形成的分生孢子梗畸形,不具有产孢能力；PdwetA的缺失突变体能够形成正常的分生孢子梗,也可以产孢,但分生孢子的细胞壁明显疏松,加厚,分生孢子色素不沉积,分生孢子萌发延迟,病菌对渗透胁迫,去垢剂和热激反应的耐受性明显下降,但对H202的耐受性却变强。qRT-PCR结果表明绿霉菌中存在PdbrlA→PdabA→PdwetA级联调控模式,而且这种模式可能存在负反馈调节机制。基因表达谱分析结果表明：与野生型相比,缺失突变体中的401个基因下调表达,144个基因上调表达。Go富集表明下调表达的基因功能主要富集在细胞膜完整性,跨膜转运和碳水化合物活性等方面,上调的基因主要参与细胞膜转运的过程。KEGG分析预测PdbrlA与淀粉和蔗糖代谢途径有关。根据烟曲霉和构巢曲霉中产孢相关基因的报道,我们在绿霉菌中找到了39个与产孢相关的同源基因。其中,12个基因(abr1、alb1、arp1、arp2、ayg1、aspf4、rodA、rodB、ppoC、axl2、 abaA和wetA)在PdbrlA中表达明显下调,2个基因(flbA和flbB)表达明显上升,12个表达下调的基因启动子区都具有brlA和abaA的转录结合位点。2.柑橘绿霉病菌通过Pdos2正调控甘油含量、负调控甾醇含量适应高盐环境高渗甘油途径在真核生物体内广泛存在,对机体适应环境变化具有极其重要的作用。Os2是该途径的一个重要激酶,柑橘绿霉病菌△PdoS2突变体对NaCl渗透压和细胞壁干扰制剂刚果红和十二烷基苯磺酸钠的敏感性显著上升,对氟咯菌腈和异菌脲两种杀菌剂的抗性也有部分提高,表明Pdos2参与了高渗透胁迫适应、细胞壁完整性和药剂抗性等生命过程。然而突变体对由H202引起的氧化压的敏感性并没有明显改变。△Pdos2突变体在接种4天后引起的病斑大小相比野生型减少了约25%,表明Pdos2对维持柑橘绿霉病菌的致病性起到部分作用。0.7M NaCl处理后,野生型菌丝体内甘油的含量明显提高而麦角甾醇的含量却显著降低。△PdoS2突变体菌丝内的甘油含量仅有轻微地上升,而麦角甾醇的含量保持与野生型相同的状态,表明Pdos2参与调节的渗透适应是与它对甘油合成的正调控和麦角甾醇合成的负调控作用有关。Pdos2基因能够直接或者间接负调控甾醇合成途径关键基因Pderg11C、Pderg1、Pderg3A/B和Pderg25等的表达。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.66
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本文编号：1355891