甘蔗应答黑穗病菌侵染的转录组与蛋白组研究及抗性相关基因挖掘
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摘要
甘蔗(Saccharum officinarum)是最重要糖料作物,蔗糖占我国食糖总产92%。病害是影响甘蔗产量和质量的重要因素,甘蔗黑穗病(Sporisorium scitamineum)是世界各植蔗区最主要的病害之一,也是我国甘蔗栽培上最重要的病害,种植抗病品种是控制该病最经济、最有效的措施。前人从相关细胞学、形态学、生理生化及抗病性遗传方面,探讨了甘蔗对黑穗病的抗性机制。目前,有关甘蔗响应黑穗病菌侵染的分子机制研究,基本还停留在以抗病甘蔗基因型接种病原后的材料进行个别几个差异表达基因的克隆与定量表达研究,这些研究初步反映了甘蔗抗黑穗病菌的分子机制受到多基因网络系统的调控,分子水平的机理与机制研究有待继续深入。利用高通量的技术手段,从转录组学和蛋白组学水平上,研究甘蔗对黑穗病菌胁迫的应答反应,有助于从整体上揭示甘蔗响应黑穗病菌侵染的代谢通路和分子调控网络,也有助于筛选与鉴定起关键作用的基因。
本研究建立了一种基于实时荧光定量PCR技术检测甘蔗黑穗病菌的方法,在黑穗病症状未出现前快速、灵敏地检测病原菌,为甘蔗黑穗病菌的快速检测和早期预防提供依据。此外,以甘蔗黑穗病抗性基因型崖城05-179和感病主栽品种"ROC”22为植物材料,通过病原菌胁迫,建立了甘蔗—黑穗病菌之间的不亲和与亲和互作系统,利用高通量RNA-Seq测序技术与iTRAQ技术,通过多点时序比较,分析甘蔗响应黑穗病病原菌胁迫过程中,同一甘蔗基因型不同接种时间点和不同甘蔗基因型不同接种时间点的mRNA及蛋白表达水平的变化,构建甘蔗应答黑穗病菌侵染的表达谱,对于从分子水平上揭示甘蔗抗黑穗病性机制具有重要的理论指导和实践意义。实验还筛选鉴定了3类抗性相关基因,包括p-1,3-葡聚糖酶基因(β-1,3-glucanase)、几丁质酶基因(chitinase)和过氧化氢酶基因(catalase),旨在为甘蔗抗病分子育种提供优异基因资源。初步研究成果如下：
1.甘蔗黑穗病是一种真菌性病害,在蔗区广泛流行,该病菌的早期诊断和准确检测将有助于该病害的治理。针对黑穗病菌bE基因(b East mating-type, GenBank登录号为U61290.1)设计特异性的实时荧光定量PCR检测引物(bEQ-F/bEQ-R)与TaqMan探针,其检出限为10ag bE质粒DNA和0.8ng甘蔗基因组DNA,比传统PCR(检出限为10fgbE质粒DNA和100ng甘蔗基因组DNA)更灵敏。实验通过检测人工接种甘蔗组培苗(FN40)进一步验证TaqMan技术的可靠性,结果显示,该技术在病原菌感染初期(12h)能够检测到黑穗病菌,适用于甘蔗无病种茎和幼苗的诊断。此外,通过不同甘蔗抗/感基因型(崖城05-179和"ROC"22)受黑穗病菌侵染后的定量检测实验,结果表明,该TaqMan技术具有进一步开发成为评价甘蔗基因型对黑穗病抗性技术的潜力。总的来说,所建立的技术体系可作为检测和诊断甘蔗黑穗病原菌的工具,实现灵敏、准确、快速和定量检测的目的。
2.以甘蔗抗黑穗病无性系崖城05-179和感黑穗病品种"ROC"22蔗芽为材料,以清水处理24h时间点为对照,对接种甘蔗黑穗病菌24h、48h和120h时间点的蔗芽进行Illumina RNA-Seq (测序)。借助维恩图(Venny diagram),通过多点时序比较,分析甘蔗响应黑穗病病原菌侵染过程中,同一基因型不同时间点和不同基因型同一时间点的差异基因表达变化,构建了甘蔗响应黑穗病病原菌侵染过程的转录组表达谱。主要结果如下：
(1)8个甘蔗样品转录组测序共获得36.68Gb数据,181,603,016对reads.分样品de novo组装后共获得148,605条Unigene,其中长度在500bp以上的有46,525条,长度在1.0kb以上的有20,798条；合并测序数据de novo组装后共获得99,824条Unigene,其中长度在500bp以上的有47,345条,长度在1.0kb以上的有22,091条。进行基于合并组装Unigene库的基因结构分析,包括ORF预测、SSR(simple sequence repeat)分析以及样品间SNP (Single Nucleotide Polymorphism)分析,获得SSR标记5,095个。将Unigene与NT、NR, SwissProt、TrEMBL、COG、GO和KEGG数据库进行比对,共获得65,852条Unigene的生物信息学注释结果。另有33,972条Unigene(34.03%)未被注释到,推测该部分序列可能是新转录本,需进一步验证。本实验构建的转录组数据库可为甘蔗其他相关研究的基因注释提供参考。
(2)进行各样品中基因表达量和差异表达基因的分析,以及对差异表达基因进行模式聚类、功能注释和富集性分析,并通过Q-PCR进行验证。结果显示：①黑穗病菌侵染后,两个甘蔗基因型被诱导的大多数差异表达基因和代谢通路都是常见的,几乎涵盖整个生命活动的各个方面。比对上的Unigene涉及细胞组件(cellular component)、分子功能(molecular function)和生物过程(biological process)等功能。在此分析基础上,获得了与抗性相关的功能注释信息,如信号转导机制(Signal transduction mechanisms)、能量产生与转导(Energy production and conversion)、离子转运机制(Inorganic ion transport and metabolism)和防御机制(Defense mechanisms)等。Pathway聚集分析显示,差异基因参与了包括植物激素信号转导途径(Plant hormone signal transductio)、类黄酮生物合成途径(Flavonoid biosynthesis)、植物与病原菌互作途径(Plant-pathogen interaction)和细胞壁防御途径(Cell wall fortification pathway)等抗病相关代谢途径。可见,黑穗病菌激发了甘蔗多种抗病途径、差异表达基因涉及防御反应及信号转导等,提示甘蔗对黑穗病病原菌侵染响应的分子机制受到多基因网络系统的调控。②随着接种黑穗病菌时间的延长,激活的甘蔗差异表达基因数目增多,且上调表达的差异基因明显多于下调表达的差异基因。黑穗病菌在崖城05-179和"ROC"22中诱导的差异基因在很大程度上是相同,但总的来说,崖城05-179中分析到的显著性抗病相关代谢通路明显多于"ROC"22,且抗性相关基因的转录表达时间(24h～48h)较"ROC"22的(48h-120h)早,此外,抗病品种中差异转录本的上／下调表达情况相对较丰富,提示它们在非亲和互作中表现抗性特异性以及较早的时序性。以上应答黑穗病菌的甘蔗转录本潜在功能的分析,有助于我们更深入研究这些候选基因在甘蔗响应黑穗病病原菌侵染中的作用,同时也为开展甘蔗抗病功能基因组学研究奠定基础。
3.在转录组学研究的基础上,以抗病和感病基因型接种黑穗病菌48h时间点的蔗芽为材料,利用iTRAQ技术,研究其蛋白组学的变化。结果表明：鉴定到的蛋白数量为4,251个,其中崖城05-179筛选到的差异表达蛋白有273个,“OC"22有341个。差异蛋白显著富集于苯丙氨酸代谢、苯丙烷类生物合成、次级代谢产物生物合成、苯并恶嗪生物合成、脂肪酸代谢和不饱和脂肪酸生物合成代谢通路中等,这些代谢通路直接或间接参与甘蔗对黑穗病菌的抗病反应。崖城05-179的蛋白组与转录组的关联性为0.1502,而"ROC"22的关联性为0.2466。甘蔗抗病和感病基因型中筛选出的与转录组关联的差异蛋白数分别为27个和10个,与差异基因表达趋势相同的差异蛋白的数量分别只有18个和10个,但均为关联上调表达,且其中近半数以上的关联差异蛋白与植物抗逆性密切相关,如过氧化物酶、病程相关蛋白PR-1、p-1,3-葡聚糖酶、热休克蛋白和植物凝集素等。研究结果为后续确定深入研究的代谢通路以及关键蛋白的鉴定提供依据。
4.甘蔗响应黑穗病菌胁迫后,在转录组测序结果中,发现病程相关蛋白(pathogenesis related proteins)的转录本差异表达,涉及10个葡聚糖酶基因(Glucanase)、26个几丁质酶基因(Chitinase)和1个过氧化氢酶基因(Catalase)。蛋白组与转录组关联分析结果也揭示了病程相关蛋白PR-1和p-1,3-葡聚糖酶等被甘蔗黑穗病菌诱导上调表达。植物病程相关蛋白是在某种病理或者与病理相关的环境条件下,植物体内诱导产生的的特异蛋白质,这些蛋白质在植物抗病反应过程中起重要作用,和植物系统获得性抗性(systemic acquired resistance, SAR)紧密相关。本研究根据该3类典型PR蛋白的差异转录本信息,借助RT-PCR技术从甘蔗中分离出2个β-1,3-葡聚糖酶基因ScGluA1和ScGluD1、1个几丁质酶基因ScChi和1过氧化氢酶基因ScCATl。通过Q-PCR技术,分析差异表达基因在黑穗病菌侵染甘蔗过程中的时空表达特性,并应用基因工程手段,进行融合蛋白亚细胞定位及目的基因遗传转化研究,为后续甘蔗抗黑穗病遗传改良提供优异基因资源。
(1)p-1,3-葡聚糖酶可以水解病原真菌细胞壁的主成分p-1,3-葡聚糖,已被证实在植物—病原菌互作中被诱导表达,且参与植物防御反应。本研究中,接种黑穗病菌后,甘蔗β-1,3-葡聚糖酶活性在抗病品种中较感病品种增加速度快且维持时间长,表明β-1,3-葡聚糖酶可能与甘蔗抗黑穗病性之间呈正相关。此外,实验从甘蔗接种黑穗病菌后的材料中,克隆到两个β-1,3-葡聚糖酶基因ScGluA1(GenBank登录号为KC848050)和ScGluD1(GenBank登录号为KC848051)。ScGluAl和ScGluD1推导的编码蛋白均存在糖苷水解酶第17家族的保守结构域,但两者之间的氨基酸序列一致性只有29.25%。系统发育树分析显示,ScGluA1和ScGluD1分别隶属于β-1,3-葡聚糖酶亚家族A和亚家族D。采用根癌农杆菌(Agrobacterium tumefaciens)介导法,将携带目标基因和报告基因(绿色荧光蛋白,GFP)的重组菌株侵染洋葱表皮细胞,结果显示ScGluA1和ScGluD1定位于细胞外或质膜上。所构建的含pET32a-ScGluA1或pET32a-ScGluD1的Rosetta (DE3)菌株原核表达细胞,在添加NaCl、CdCl2、PEG、CuCl2或ZnS04的培养基中,表现出不同的耐受性。Q-PCR分析结果表明,ScGluA1在应答黑穗病菌中上调表达, ScGluD1基因小幅下调表达,暗示ScGluA1可能涉及甘蔗对黑穗病菌的防御反应,而ScGluD1可能被抑制。受其他环境因子(如水杨酸、茉莉酸甲酯、脱落酸、NaCl、CdCl2和自然干旱胁迫)处理后,ScGluA1和ScGluD1的基因表达模式与生物胁迫类似。在烟草中过表达ScGluA1,对烟草茄病镰刀菌蓝色变种(Fusarium solani var. coeruleum)和烟草灰葡萄孢菌(Botrytis cinerea)有抑制效果,且转ScGluA1烟草T0代植株叶片β-1,3-葡聚糖酶粗酶液,对烟草茄病镰刀菌蓝色变种的菌丝生长有一定的抑制作用。总的来说,β-1,3-葡聚糖酶可能作为甘蔗对黑穗病菌防御机制中的一个组分。ScGluA1的积极应答或许对甘蔗抗病性和抗逆反应有益,而ScGluD1的消极应答,显示其在甘蔗响应生物和非生物胁迫中,可能发挥不同的作用。
(2)许多植物与病原菌互作会诱导产生几丁质酶(EC3.2.2.14),报道显示其与植物防御病原菌相关。本研究发现,几丁质酶活性与甘蔗黑穗病抗性之间呈正相关。实验克隆了一个Class Ⅲ几丁质酶基因ScChi (GenBank登录号为KF664180),编码一个31.37kDa多肽。亚细胞定位显示,ScChi定位于细胞核、细胞质和质膜上。Q-PCR结果表明,ScChi在叶和蔗皮组织中高度表达。受黑穗病菌侵染后,ScChi转录水平在抗病品种中较感病品种高,且维持时间较长。ScChi同时受SA、H2O2、MeJA、ABA、 NaCl、CuCl2、PEG和低温(4℃)胁迫的诱导表达。ScChi瞬时表达后的目标基因的转录水平及6个本氏烟(Nicotiana benthamiana)免疫相关标记基因均上调,而与对照相比,过表达pCAMBIA1301-ScChi的本氏烟叶片经组织化学分析,结果显示出更深的DAB染色效果及更高的导电率,表明存在高水平的H2O2积累。此外,瞬时表达ScChi有助于提高烟草对青枯菌(Pseudomonas solanacearum)、茄病镰刀菌蓝色变种和灰葡萄孢菌的防御作用,同时,转ScChi烟草T0代植株叶片的几丁质酶粗酶液,可以抑制茄病镰刀菌蓝色变种的菌丝生长。这些结果暗示,ScChi的表达与植物免疫之间关系密切。总的来说,ScChi能积极响应生物和非生物胁迫,表明该基因在甘蔗中是一类与逆境胁迫相关的抗病基因。
(3)过氧化氢酶是铁卟啉酶,它作为一种有效的活性氧(Reactive Oxygen Species, ROS)清除剂,可以保护植物避免氧化损伤。过氧化氢酶的酶活性在甘蔗抗病品种(崖城05-179)应答黑穗病菌中总是高于感病品种(柳城03-182),表明过氧化氢酶活性可能与甘蔗黑穗病抗性呈正相关。为了从分子水平上了解过氧化氢酶的功能,实验从受黑穗病菌侵染的甘蔗材料中,分离到一个ScCAT1(GenBank登录号为KF664183)的cDNA序列。该基因预测编码492个氨基酸残基,其推导的蛋白序列与其他植物的过氧化氢酶同源性高。ScCAT1的重组大肠杆菌Rosetta细胞,在CuCl、CdCl2和NaCl的胁迫下长势更快,表明其耐受能力更强。Q-PCR分析结果显示,ScCAT1在蔗芽中的表达量相对较高,而在蔗皮和蔗髓的表达量中等。不同外源胁迫包括甘蔗黑穗病菌侵染、植物激素(SA、MeJA和ABA)处理、氧化胁迫(H2O2)、重金属(CuCl2)和高渗透(PEG和NaCl)胁迫,均诱导ScCAT1基因上调表达。亚细胞定位结果显示,ScCAT1定位在质膜和细胞质。此外,DAB、台盼蓝染色和电导率测定等组织化学分析表明,ScCAT1基因可能涉及甘蔗的免疫应答。总的来说,ScCAT1正响应生物和非生物胁迫的研究结果表明,ScCAT1参与保护甘蔗应答与氧化反应相关的环境刺激。
Sugarcane (Saccharum officinarum) is the most important sugar crop in the worldwide, the sucrose accounted for92%of total sugar output in China. Plant disease is an important factor that affects the sugarcane yield and quality. Sugarcane smut (Sporisorium scitamineum) is one of the main diseases in the sugarcane planting area. It is generally believed that using sugarcane disease-resistant cultivar instead of susceptible one is the most economic and effective measure to prevent smut. At present, the related researches focus on the studies of cytology, morphology, physiology and biochemistry, as well as genetics, to explore the resistant mechanism to smut in sugarcane. While the research on the interaction between sugarcane and S. scitamineum at the molecular level still focuses on the cloning and quantification of a few resistance-related genes in the sugarcane genotypes after inoculation with S. scitamineum. These preliminary researches reflect the molecular mechanism of sugarcane resistant to S. scitamineum is regulated by multiple gene networks. But the above studies remain to be further. Using transcriptomics and proteomics to explore the impact of S. scitamineum on sugarcane can help to reveal the metabolic pathways, the molecular regulation networks and key genes during sugarcane response to S. scitamineum.
In this study, a new detection method based on real-time fluorescent quantitative PCR (Q-PCR) was established, which could detect S. scitamineum rapidly and sensitively before disease symptom appear. It provides a scientific basis for rapidly detect and prevent S. scitamineum earlier. In this study, two sugarcane genotypes, one is an intergeneric BC2hybrid with good resistance characters named YachengO5-179from S. spp. hybrid X S. arundinaceum, the other is the present major cultivar "ROC"22which was susceptible to S. scitamineum, were taken as plant materials to establish affinity and the affinity interaction systems with S. scitamineum. The aim of this study was to reveal the molecular mechanism related to metabolism and signaling network of sugarcane response to S. scitamineum stress on the whole level through the RNA-Seq and iTRAQ technologies. Through multi-point time series comparison, the change of mRNA expression level was analysed. And the transcriptome expression profile was built in different time points in both sugarcane genotypes after inoculation with S. scitamineum. Then, three types of resistance-related genes, including P-l,3-glucanase, chitinase and catalase were mined, which can provide new gene resources for sugarcane breeding resistant to fungal diseases. The main results and conclusions are as follows.
1. Smut is a fungal disease which widespread popularity in sugarcane planting area. Early detection and proper identification of S. scitamineum are essential in smut management practice. Here, we developed a TaqMan real-time PCR assay using specific primers (bEQ-F/bEQ-R) and a TaqMan probe (bEQ-P) which were designed based on the bE (b East mating-type) gene (GenBank Accession No. U61290.1). This method was more sensitive (a detection limit of10ag pbE DNA and0.8ng sugarcane genomic DNA) than that of conventional PCR (10fg and100ng, respectively). Reliability was demonstrated through the positive detection of samples collected from artificially-inoculated sugarcane plantlets (FN40). This assay was capable of detecting the smut pathogen at the initial stage (12h) of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. Furthermore, quantification of pathogen was verified in pathogen challenged buds in different sugarcane genotypes (Yacheng05-179and "ROC"22), which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. Taken together, this novel assay can be used as a diagnostic tool for sensitive, accurate, fast and quantitative detection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcane genotypes.
2. Samples of Yacheng05-179(resistance) and "ROC'22(susceptible) inoculated with sterile water for24h (control) and S. scitamineum for24h,48h and120h were used to analyze the transcriptome by Illumina RNA-Seq. The results showed that:
(1) In total,36.68Gb data and181,603,016reads were generated from eight sugarcane samples through transcriptome sequencing. The total of148,605Unigenes were obtained by de novo assembly in separate samples, which contained46,525Unigenes at length of more than500bp and20,798Unigenes at length of more than1.0kb, while both Unigene were reduced to47,345and22,091after merging. Based on the Merge_assembly Unigene library, the gene structure was analyzed, including ORF prediction, simple sequence repeats (SSR) analysis and single nucleotide polymorphisms (SNP) analysis. Then,5,095SSR markers were generated. Comparing Unigene with NT, NR, SwissProt, TrEMBL, COG, GO and KEGG databases, bioinformatics annotation results of65,852Unigene were found. Besides,33,972Unigene (34.03%) had not been annotated. We speculate that these sequences without annotation may be new transcripts, which need further verification. The transcriptome database built in this study can provide reference for other related studies in sugarcane.
(2) The analysis of gene expression quantity and the differential expression genes in each sample were performed, as well as the gene clustering pattern, functional annotation and enrichment analysis. The candidate genes were verified by Q-PCR method. The results suggest that:①The induction of the majority of the differential expression genes and metabolic pathways were common in the two sugarcane cultivars after challenge by S. scitamineum. Almost of them referred to all aspects of the life activities. These Unigene were involved in molecular functions, cellular components and biological process. The resistance related functional annotation was obtained, such as signal transduction mechanisms, energy production and conversion, inorganic ion transport, metabolism and defense mechanisms, etc. Pathway clustering analysis showed that, the differential expression genes involved in resistance related metabolic pathways of plant hormone signal transduction, flavonoid biosynthesis, plant-pathogen interaction and the cell wall fortification pathway, etc. Visibility, various of disease resistance pathways and the differential expression genes which referred to defense reaction and signal transduction were inspired in sugarcane after inoculation with S. scitamineum. These reveal that the molecular mechanism of response to smut pathogen infection was controlled by polygenic network system.②As the extension of inoculational time after S. scitamineum infection, the number of sugarcane differential expression genes were increased. What's more, the total number of the up-regulated genes were significantly more than that of the down-regulated one. Although differential expression genes in YachengO5-179and "ROC"22were largely the same, the significant resistance related metabolic pathways were more than that of in "ROC"22. Meanwhile, the resistance related genes were expressed earlier (24h-48h) in YachengO5-179than that of in "ROC"22(48h-120h). And the gene expression pattern of up/down-regulation was more abundant in the resistant genotype. It reveals a resistance of specificity or earlier scheduling is performed in the affinity interaction. The potential function analysis of the above transcripts in sugarcane response to S. scitamineum can help us understand the role of these candidate genes in sugarcane response to smut pathogen infection, as well as in the study of the functional genomics of sugarcane disease resistance.
3. On the basis of the transcriptome study, change of proteomics of YachengO5-179and "ROC"22inoculated with S. scitamineum after48h was analysed by iTRAQ technology. It showed that the number of identified proteins was4,251, including273differential expression proteins in YachengO5-179and341in "ROC"22. These differential expression proteins were significantly involved in phenylalanine metabolism, phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, benzoxazinoid biosynthesis, fatty acid metabolism and biosynthesis of unsaturated fatty acids. The correlation analyses showed that after infection with S. scitamineum for48h, the correlations between the proteomics and transcriptomes were0.1502and0.2466in Yacheng05-179and "ROC"22, respectively. Twenty seven and ten differential expression proteins associated with the transcriptome were selected in the resistant variety and susceptible one, respectively. And the number of the differential expression proteins which were expressed at the same trend with differential expression genes were18and10, respectively. Most differential proteins were closely related to plant's stress resistance, such as peroxidase, pathogenesis-related protein, beta-1,3-glucanase, heat shock protein and lectin. These results provide the basis for the determination of the subsequent metabolic pathways and the identification of key proteins.
4. The transcripts of pathogenesis related proteins were differentially expressed after inoculated with S. scitamineum, involving10glucanase genes,26chitinase genes and1catalase gene. Studies on proteome associated with transcriptome showed pathogenesis-related protein1and beta-1,-3-glucanase are induced by S. scitamineum. As we know, pathogenesis related proteins are specific proteins induced by a sort of pathological conditions. They play an important role in the process of plant disease resistance reaction, and are closely related to systemic acquired resistance (SAR). According to the differential expression transcripts of these three kinds of typical pathogenesis related proteins, two β-1,3-glucanase genes ScGluAl and ScGluDl, one chitinase gene ScChi and one catalase gene ScCAT1were isolated from sugarcane by RT-PCR assay. The character of the spatio-temporal expression in differential expression genes was elucidated by Q-PCR. Subcellular location of fusion protein and the genetic transformation research of the target genes were applied by genetic engineering technology. This work will contribute to further genetic improvement of smut resistance in sugarcane by providing the excellent gene resources or technique.
(1) β-1,3-glucanase can hydrolyze the main component of fungal cell wall of β-1,3-glucan, has been shown to express during plant-pathogen interaction and involved in plant defense response. In this study, β-1,3-glucanase enzyme activity in the resistant cultivar increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1(GenBank Accession No. KC848050) and ScGluD1(GenBank Accession No. KC848051), were cloned and characterized. Predicted amino acid sequences viz both ScGluAl and ScGluDl genes contain a glycosyl hydrolases family17domain, between which only29.25%consistency at the amino acid sequence level. Phylogenetic analysis suggested that ScGluAl and ScGluDl clustered within subfamily A and subfamily D, respectively. Agrobacterium-mediated transformation for transient expression of target genes (ScGluAl and ScGluDl) with green fluorescent protein (GFP) in onion epidermal peels demonstrated that both gene products were targeted to apoplast. Escherich coli Rosetta (DE3) cells expressing ScGluA1and ScGluD1showed varying degrees of tolerance to NaCl, CdCl2, PEG, CuCl2and ZnSO4·Q-PCR analysis showed up-regulation of ScGluA1and slight down-regulation of ScGluDl in response to S. scitamineum infection. It suggested that ScGluA1may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1was inhibited. Gene expression patterns of ScGluA1and ScGluDl, in response to other factors such as salicylic acid (SA), methyl jasmonate (MeJA), abscissic acid (ABA), NaCl, CdCl2and natural drought stresses, were similar to sugarcane response against smut infection. Over expression of ScGluA1in Nicotiana benthamiana showed an antimicrobial action on Fusarium solani var. coeruleum and Botrytis cinerea. Moreover, β-1,3-glucanase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. Together, β-1,3-glucanase may work as a component in sugarcane defense mechanisms for S. scitamineum. The positive responses of ScGluAl may contribute sugarcane resistance to adversity stress; while the negative responses of ScGluDl to biotic and abiotic stresses indicate it plays a different role from that of ScGluA1in interaction between sugarcane and biotic or abiotic stresses.
(2) Chitinases (EC3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank Accession No. KF664180), a Class III chitinase gene encoded a31.37kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Q-PCR results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with S. scitamineum. The ScChi also showed obvious induction of transcription after treatment with SA, H2O2, MeJA, ABA, NaCl, CuCl2, PEG and low temperature (4℃). The expression levels of ScChi and six immunity associate marker genes were up-regulated by the transient over expression of ScChi. The histochemical assay of Nicotiana benthamiana leaves over-expressing pCAMBIA1301-ScChi exhibited deep DAB staining color and high conductivity, indicating the high level of H2O2accumulation. Besides, over-expressing ScChi helped improve N. benthamiana to defense Pseudomonas solanacearum, Fusarium solani var. coeruleum and Botrytis cinerea. Meanwhile, chitinase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.
(3) Catalase is an iron porphyrin enzyme, which serves as an efficient scavenger of reactive oxygen species (ROS) to avoid oxidative damage. In sugarcane, the enzymatic activity of catalase in the cultivar (Yacheng05-179) resistant to the smut pathogen S. scitamineum was always higher than that of the susceptible cultivar (Liucheng03-182), suggesting that catalase activity may have a positive correlation with smut resistance in sugarcane. To understand the function of catalase at the molecular level, a cDNA sequence of ScCAT1(GenBank Accession No. KF664183), was isolated from sugarcane infected by S. scitamineum. ScCAT1was predicted to encode492amino acid residues, and its deduced amino acid sequence shared a high degree of homology with other plant catalases. Enhanced growth of ScCAT1in recombinant Escherichia coli Rosetta cells under the stresses of CuCl2, CdCl2and NaCl indicated its high tolerance. Q-PCR results showed that ScCATl was expressed at relatively high levels in the bud, whereas expression was moderate in stem epidermis and stem pith. Different kinds of stresses, including S. scitamineum challenge, plant hormones (SA, MeJA and ABA) treatments, oxidative (H2O2) stress, heavy metal (CuCl2) and hyper-osmotic (PEG and NaC1) stresses, triggered a significant induction of ScCATl. The ScCAT1protein appeared to localize in plasma membrane and cytoplasm. Furthermore, histochemical assays using DAB and trypan blue staining, as well as conductivity measurement, indicated that ScCAT1may confer the sugarcane immunity. In conclusion, the positive response of ScCAT1to biotic and abiotic stresses suggests that ScCAT1is involved in protection of sugarcane against reactive oxidant-related environmental stimuli.
本研究建立了一种基于实时荧光定量PCR技术检测甘蔗黑穗病菌的方法,在黑穗病症状未出现前快速、灵敏地检测病原菌,为甘蔗黑穗病菌的快速检测和早期预防提供依据。此外,以甘蔗黑穗病抗性基因型崖城05-179和感病主栽品种"ROC”22为植物材料,通过病原菌胁迫,建立了甘蔗—黑穗病菌之间的不亲和与亲和互作系统,利用高通量RNA-Seq测序技术与iTRAQ技术,通过多点时序比较,分析甘蔗响应黑穗病病原菌胁迫过程中,同一甘蔗基因型不同接种时间点和不同甘蔗基因型不同接种时间点的mRNA及蛋白表达水平的变化,构建甘蔗应答黑穗病菌侵染的表达谱,对于从分子水平上揭示甘蔗抗黑穗病性机制具有重要的理论指导和实践意义。实验还筛选鉴定了3类抗性相关基因,包括p-1,3-葡聚糖酶基因(β-1,3-glucanase)、几丁质酶基因(chitinase)和过氧化氢酶基因(catalase),旨在为甘蔗抗病分子育种提供优异基因资源。初步研究成果如下：
1.甘蔗黑穗病是一种真菌性病害,在蔗区广泛流行,该病菌的早期诊断和准确检测将有助于该病害的治理。针对黑穗病菌bE基因(b East mating-type, GenBank登录号为U61290.1)设计特异性的实时荧光定量PCR检测引物(bEQ-F/bEQ-R)与TaqMan探针,其检出限为10ag bE质粒DNA和0.8ng甘蔗基因组DNA,比传统PCR(检出限为10fgbE质粒DNA和100ng甘蔗基因组DNA)更灵敏。实验通过检测人工接种甘蔗组培苗(FN40)进一步验证TaqMan技术的可靠性,结果显示,该技术在病原菌感染初期(12h)能够检测到黑穗病菌,适用于甘蔗无病种茎和幼苗的诊断。此外,通过不同甘蔗抗/感基因型(崖城05-179和"ROC"22)受黑穗病菌侵染后的定量检测实验,结果表明,该TaqMan技术具有进一步开发成为评价甘蔗基因型对黑穗病抗性技术的潜力。总的来说,所建立的技术体系可作为检测和诊断甘蔗黑穗病原菌的工具,实现灵敏、准确、快速和定量检测的目的。
2.以甘蔗抗黑穗病无性系崖城05-179和感黑穗病品种"ROC"22蔗芽为材料,以清水处理24h时间点为对照,对接种甘蔗黑穗病菌24h、48h和120h时间点的蔗芽进行Illumina RNA-Seq (测序)。借助维恩图(Venny diagram),通过多点时序比较,分析甘蔗响应黑穗病病原菌侵染过程中,同一基因型不同时间点和不同基因型同一时间点的差异基因表达变化,构建了甘蔗响应黑穗病病原菌侵染过程的转录组表达谱。主要结果如下：
(1)8个甘蔗样品转录组测序共获得36.68Gb数据,181,603,016对reads.分样品de novo组装后共获得148,605条Unigene,其中长度在500bp以上的有46,525条,长度在1.0kb以上的有20,798条；合并测序数据de novo组装后共获得99,824条Unigene,其中长度在500bp以上的有47,345条,长度在1.0kb以上的有22,091条。进行基于合并组装Unigene库的基因结构分析,包括ORF预测、SSR(simple sequence repeat)分析以及样品间SNP (Single Nucleotide Polymorphism)分析,获得SSR标记5,095个。将Unigene与NT、NR, SwissProt、TrEMBL、COG、GO和KEGG数据库进行比对,共获得65,852条Unigene的生物信息学注释结果。另有33,972条Unigene(34.03%)未被注释到,推测该部分序列可能是新转录本,需进一步验证。本实验构建的转录组数据库可为甘蔗其他相关研究的基因注释提供参考。
(2)进行各样品中基因表达量和差异表达基因的分析,以及对差异表达基因进行模式聚类、功能注释和富集性分析,并通过Q-PCR进行验证。结果显示：①黑穗病菌侵染后,两个甘蔗基因型被诱导的大多数差异表达基因和代谢通路都是常见的,几乎涵盖整个生命活动的各个方面。比对上的Unigene涉及细胞组件(cellular component)、分子功能(molecular function)和生物过程(biological process)等功能。在此分析基础上,获得了与抗性相关的功能注释信息,如信号转导机制(Signal transduction mechanisms)、能量产生与转导(Energy production and conversion)、离子转运机制(Inorganic ion transport and metabolism)和防御机制(Defense mechanisms)等。Pathway聚集分析显示,差异基因参与了包括植物激素信号转导途径(Plant hormone signal transductio)、类黄酮生物合成途径(Flavonoid biosynthesis)、植物与病原菌互作途径(Plant-pathogen interaction)和细胞壁防御途径(Cell wall fortification pathway)等抗病相关代谢途径。可见,黑穗病菌激发了甘蔗多种抗病途径、差异表达基因涉及防御反应及信号转导等,提示甘蔗对黑穗病病原菌侵染响应的分子机制受到多基因网络系统的调控。②随着接种黑穗病菌时间的延长,激活的甘蔗差异表达基因数目增多,且上调表达的差异基因明显多于下调表达的差异基因。黑穗病菌在崖城05-179和"ROC"22中诱导的差异基因在很大程度上是相同,但总的来说,崖城05-179中分析到的显著性抗病相关代谢通路明显多于"ROC"22,且抗性相关基因的转录表达时间(24h～48h)较"ROC"22的(48h-120h)早,此外,抗病品种中差异转录本的上／下调表达情况相对较丰富,提示它们在非亲和互作中表现抗性特异性以及较早的时序性。以上应答黑穗病菌的甘蔗转录本潜在功能的分析,有助于我们更深入研究这些候选基因在甘蔗响应黑穗病病原菌侵染中的作用,同时也为开展甘蔗抗病功能基因组学研究奠定基础。
3.在转录组学研究的基础上,以抗病和感病基因型接种黑穗病菌48h时间点的蔗芽为材料,利用iTRAQ技术,研究其蛋白组学的变化。结果表明：鉴定到的蛋白数量为4,251个,其中崖城05-179筛选到的差异表达蛋白有273个,“OC"22有341个。差异蛋白显著富集于苯丙氨酸代谢、苯丙烷类生物合成、次级代谢产物生物合成、苯并恶嗪生物合成、脂肪酸代谢和不饱和脂肪酸生物合成代谢通路中等,这些代谢通路直接或间接参与甘蔗对黑穗病菌的抗病反应。崖城05-179的蛋白组与转录组的关联性为0.1502,而"ROC"22的关联性为0.2466。甘蔗抗病和感病基因型中筛选出的与转录组关联的差异蛋白数分别为27个和10个,与差异基因表达趋势相同的差异蛋白的数量分别只有18个和10个,但均为关联上调表达,且其中近半数以上的关联差异蛋白与植物抗逆性密切相关,如过氧化物酶、病程相关蛋白PR-1、p-1,3-葡聚糖酶、热休克蛋白和植物凝集素等。研究结果为后续确定深入研究的代谢通路以及关键蛋白的鉴定提供依据。
4.甘蔗响应黑穗病菌胁迫后,在转录组测序结果中,发现病程相关蛋白(pathogenesis related proteins)的转录本差异表达,涉及10个葡聚糖酶基因(Glucanase)、26个几丁质酶基因(Chitinase)和1个过氧化氢酶基因(Catalase)。蛋白组与转录组关联分析结果也揭示了病程相关蛋白PR-1和p-1,3-葡聚糖酶等被甘蔗黑穗病菌诱导上调表达。植物病程相关蛋白是在某种病理或者与病理相关的环境条件下,植物体内诱导产生的的特异蛋白质,这些蛋白质在植物抗病反应过程中起重要作用,和植物系统获得性抗性(systemic acquired resistance, SAR)紧密相关。本研究根据该3类典型PR蛋白的差异转录本信息,借助RT-PCR技术从甘蔗中分离出2个β-1,3-葡聚糖酶基因ScGluA1和ScGluD1、1个几丁质酶基因ScChi和1过氧化氢酶基因ScCATl。通过Q-PCR技术,分析差异表达基因在黑穗病菌侵染甘蔗过程中的时空表达特性,并应用基因工程手段,进行融合蛋白亚细胞定位及目的基因遗传转化研究,为后续甘蔗抗黑穗病遗传改良提供优异基因资源。
(1)p-1,3-葡聚糖酶可以水解病原真菌细胞壁的主成分p-1,3-葡聚糖,已被证实在植物—病原菌互作中被诱导表达,且参与植物防御反应。本研究中,接种黑穗病菌后,甘蔗β-1,3-葡聚糖酶活性在抗病品种中较感病品种增加速度快且维持时间长,表明β-1,3-葡聚糖酶可能与甘蔗抗黑穗病性之间呈正相关。此外,实验从甘蔗接种黑穗病菌后的材料中,克隆到两个β-1,3-葡聚糖酶基因ScGluA1(GenBank登录号为KC848050)和ScGluD1(GenBank登录号为KC848051)。ScGluAl和ScGluD1推导的编码蛋白均存在糖苷水解酶第17家族的保守结构域,但两者之间的氨基酸序列一致性只有29.25%。系统发育树分析显示,ScGluA1和ScGluD1分别隶属于β-1,3-葡聚糖酶亚家族A和亚家族D。采用根癌农杆菌(Agrobacterium tumefaciens)介导法,将携带目标基因和报告基因(绿色荧光蛋白,GFP)的重组菌株侵染洋葱表皮细胞,结果显示ScGluA1和ScGluD1定位于细胞外或质膜上。所构建的含pET32a-ScGluA1或pET32a-ScGluD1的Rosetta (DE3)菌株原核表达细胞,在添加NaCl、CdCl2、PEG、CuCl2或ZnS04的培养基中,表现出不同的耐受性。Q-PCR分析结果表明,ScGluA1在应答黑穗病菌中上调表达, ScGluD1基因小幅下调表达,暗示ScGluA1可能涉及甘蔗对黑穗病菌的防御反应,而ScGluD1可能被抑制。受其他环境因子(如水杨酸、茉莉酸甲酯、脱落酸、NaCl、CdCl2和自然干旱胁迫)处理后,ScGluA1和ScGluD1的基因表达模式与生物胁迫类似。在烟草中过表达ScGluA1,对烟草茄病镰刀菌蓝色变种(Fusarium solani var. coeruleum)和烟草灰葡萄孢菌(Botrytis cinerea)有抑制效果,且转ScGluA1烟草T0代植株叶片β-1,3-葡聚糖酶粗酶液,对烟草茄病镰刀菌蓝色变种的菌丝生长有一定的抑制作用。总的来说,β-1,3-葡聚糖酶可能作为甘蔗对黑穗病菌防御机制中的一个组分。ScGluA1的积极应答或许对甘蔗抗病性和抗逆反应有益,而ScGluD1的消极应答,显示其在甘蔗响应生物和非生物胁迫中,可能发挥不同的作用。
(2)许多植物与病原菌互作会诱导产生几丁质酶(EC3.2.2.14),报道显示其与植物防御病原菌相关。本研究发现,几丁质酶活性与甘蔗黑穗病抗性之间呈正相关。实验克隆了一个Class Ⅲ几丁质酶基因ScChi (GenBank登录号为KF664180),编码一个31.37kDa多肽。亚细胞定位显示,ScChi定位于细胞核、细胞质和质膜上。Q-PCR结果表明,ScChi在叶和蔗皮组织中高度表达。受黑穗病菌侵染后,ScChi转录水平在抗病品种中较感病品种高,且维持时间较长。ScChi同时受SA、H2O2、MeJA、ABA、 NaCl、CuCl2、PEG和低温(4℃)胁迫的诱导表达。ScChi瞬时表达后的目标基因的转录水平及6个本氏烟(Nicotiana benthamiana)免疫相关标记基因均上调,而与对照相比,过表达pCAMBIA1301-ScChi的本氏烟叶片经组织化学分析,结果显示出更深的DAB染色效果及更高的导电率,表明存在高水平的H2O2积累。此外,瞬时表达ScChi有助于提高烟草对青枯菌(Pseudomonas solanacearum)、茄病镰刀菌蓝色变种和灰葡萄孢菌的防御作用,同时,转ScChi烟草T0代植株叶片的几丁质酶粗酶液,可以抑制茄病镰刀菌蓝色变种的菌丝生长。这些结果暗示,ScChi的表达与植物免疫之间关系密切。总的来说,ScChi能积极响应生物和非生物胁迫,表明该基因在甘蔗中是一类与逆境胁迫相关的抗病基因。
(3)过氧化氢酶是铁卟啉酶,它作为一种有效的活性氧(Reactive Oxygen Species, ROS)清除剂,可以保护植物避免氧化损伤。过氧化氢酶的酶活性在甘蔗抗病品种(崖城05-179)应答黑穗病菌中总是高于感病品种(柳城03-182),表明过氧化氢酶活性可能与甘蔗黑穗病抗性呈正相关。为了从分子水平上了解过氧化氢酶的功能,实验从受黑穗病菌侵染的甘蔗材料中,分离到一个ScCAT1(GenBank登录号为KF664183)的cDNA序列。该基因预测编码492个氨基酸残基,其推导的蛋白序列与其他植物的过氧化氢酶同源性高。ScCAT1的重组大肠杆菌Rosetta细胞,在CuCl、CdCl2和NaCl的胁迫下长势更快,表明其耐受能力更强。Q-PCR分析结果显示,ScCAT1在蔗芽中的表达量相对较高,而在蔗皮和蔗髓的表达量中等。不同外源胁迫包括甘蔗黑穗病菌侵染、植物激素(SA、MeJA和ABA)处理、氧化胁迫(H2O2)、重金属(CuCl2)和高渗透(PEG和NaCl)胁迫,均诱导ScCAT1基因上调表达。亚细胞定位结果显示,ScCAT1定位在质膜和细胞质。此外,DAB、台盼蓝染色和电导率测定等组织化学分析表明,ScCAT1基因可能涉及甘蔗的免疫应答。总的来说,ScCAT1正响应生物和非生物胁迫的研究结果表明,ScCAT1参与保护甘蔗应答与氧化反应相关的环境刺激。
Sugarcane (Saccharum officinarum) is the most important sugar crop in the worldwide, the sucrose accounted for92%of total sugar output in China. Plant disease is an important factor that affects the sugarcane yield and quality. Sugarcane smut (Sporisorium scitamineum) is one of the main diseases in the sugarcane planting area. It is generally believed that using sugarcane disease-resistant cultivar instead of susceptible one is the most economic and effective measure to prevent smut. At present, the related researches focus on the studies of cytology, morphology, physiology and biochemistry, as well as genetics, to explore the resistant mechanism to smut in sugarcane. While the research on the interaction between sugarcane and S. scitamineum at the molecular level still focuses on the cloning and quantification of a few resistance-related genes in the sugarcane genotypes after inoculation with S. scitamineum. These preliminary researches reflect the molecular mechanism of sugarcane resistant to S. scitamineum is regulated by multiple gene networks. But the above studies remain to be further. Using transcriptomics and proteomics to explore the impact of S. scitamineum on sugarcane can help to reveal the metabolic pathways, the molecular regulation networks and key genes during sugarcane response to S. scitamineum.
In this study, a new detection method based on real-time fluorescent quantitative PCR (Q-PCR) was established, which could detect S. scitamineum rapidly and sensitively before disease symptom appear. It provides a scientific basis for rapidly detect and prevent S. scitamineum earlier. In this study, two sugarcane genotypes, one is an intergeneric BC2hybrid with good resistance characters named YachengO5-179from S. spp. hybrid X S. arundinaceum, the other is the present major cultivar "ROC"22which was susceptible to S. scitamineum, were taken as plant materials to establish affinity and the affinity interaction systems with S. scitamineum. The aim of this study was to reveal the molecular mechanism related to metabolism and signaling network of sugarcane response to S. scitamineum stress on the whole level through the RNA-Seq and iTRAQ technologies. Through multi-point time series comparison, the change of mRNA expression level was analysed. And the transcriptome expression profile was built in different time points in both sugarcane genotypes after inoculation with S. scitamineum. Then, three types of resistance-related genes, including P-l,3-glucanase, chitinase and catalase were mined, which can provide new gene resources for sugarcane breeding resistant to fungal diseases. The main results and conclusions are as follows.
1. Smut is a fungal disease which widespread popularity in sugarcane planting area. Early detection and proper identification of S. scitamineum are essential in smut management practice. Here, we developed a TaqMan real-time PCR assay using specific primers (bEQ-F/bEQ-R) and a TaqMan probe (bEQ-P) which were designed based on the bE (b East mating-type) gene (GenBank Accession No. U61290.1). This method was more sensitive (a detection limit of10ag pbE DNA and0.8ng sugarcane genomic DNA) than that of conventional PCR (10fg and100ng, respectively). Reliability was demonstrated through the positive detection of samples collected from artificially-inoculated sugarcane plantlets (FN40). This assay was capable of detecting the smut pathogen at the initial stage (12h) of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. Furthermore, quantification of pathogen was verified in pathogen challenged buds in different sugarcane genotypes (Yacheng05-179and "ROC"22), which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. Taken together, this novel assay can be used as a diagnostic tool for sensitive, accurate, fast and quantitative detection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcane genotypes.
2. Samples of Yacheng05-179(resistance) and "ROC'22(susceptible) inoculated with sterile water for24h (control) and S. scitamineum for24h,48h and120h were used to analyze the transcriptome by Illumina RNA-Seq. The results showed that:
(1) In total,36.68Gb data and181,603,016reads were generated from eight sugarcane samples through transcriptome sequencing. The total of148,605Unigenes were obtained by de novo assembly in separate samples, which contained46,525Unigenes at length of more than500bp and20,798Unigenes at length of more than1.0kb, while both Unigene were reduced to47,345and22,091after merging. Based on the Merge_assembly Unigene library, the gene structure was analyzed, including ORF prediction, simple sequence repeats (SSR) analysis and single nucleotide polymorphisms (SNP) analysis. Then,5,095SSR markers were generated. Comparing Unigene with NT, NR, SwissProt, TrEMBL, COG, GO and KEGG databases, bioinformatics annotation results of65,852Unigene were found. Besides,33,972Unigene (34.03%) had not been annotated. We speculate that these sequences without annotation may be new transcripts, which need further verification. The transcriptome database built in this study can provide reference for other related studies in sugarcane.
(2) The analysis of gene expression quantity and the differential expression genes in each sample were performed, as well as the gene clustering pattern, functional annotation and enrichment analysis. The candidate genes were verified by Q-PCR method. The results suggest that:①The induction of the majority of the differential expression genes and metabolic pathways were common in the two sugarcane cultivars after challenge by S. scitamineum. Almost of them referred to all aspects of the life activities. These Unigene were involved in molecular functions, cellular components and biological process. The resistance related functional annotation was obtained, such as signal transduction mechanisms, energy production and conversion, inorganic ion transport, metabolism and defense mechanisms, etc. Pathway clustering analysis showed that, the differential expression genes involved in resistance related metabolic pathways of plant hormone signal transduction, flavonoid biosynthesis, plant-pathogen interaction and the cell wall fortification pathway, etc. Visibility, various of disease resistance pathways and the differential expression genes which referred to defense reaction and signal transduction were inspired in sugarcane after inoculation with S. scitamineum. These reveal that the molecular mechanism of response to smut pathogen infection was controlled by polygenic network system.②As the extension of inoculational time after S. scitamineum infection, the number of sugarcane differential expression genes were increased. What's more, the total number of the up-regulated genes were significantly more than that of the down-regulated one. Although differential expression genes in YachengO5-179and "ROC"22were largely the same, the significant resistance related metabolic pathways were more than that of in "ROC"22. Meanwhile, the resistance related genes were expressed earlier (24h-48h) in YachengO5-179than that of in "ROC"22(48h-120h). And the gene expression pattern of up/down-regulation was more abundant in the resistant genotype. It reveals a resistance of specificity or earlier scheduling is performed in the affinity interaction. The potential function analysis of the above transcripts in sugarcane response to S. scitamineum can help us understand the role of these candidate genes in sugarcane response to smut pathogen infection, as well as in the study of the functional genomics of sugarcane disease resistance.
3. On the basis of the transcriptome study, change of proteomics of YachengO5-179and "ROC"22inoculated with S. scitamineum after48h was analysed by iTRAQ technology. It showed that the number of identified proteins was4,251, including273differential expression proteins in YachengO5-179and341in "ROC"22. These differential expression proteins were significantly involved in phenylalanine metabolism, phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, benzoxazinoid biosynthesis, fatty acid metabolism and biosynthesis of unsaturated fatty acids. The correlation analyses showed that after infection with S. scitamineum for48h, the correlations between the proteomics and transcriptomes were0.1502and0.2466in Yacheng05-179and "ROC"22, respectively. Twenty seven and ten differential expression proteins associated with the transcriptome were selected in the resistant variety and susceptible one, respectively. And the number of the differential expression proteins which were expressed at the same trend with differential expression genes were18and10, respectively. Most differential proteins were closely related to plant's stress resistance, such as peroxidase, pathogenesis-related protein, beta-1,3-glucanase, heat shock protein and lectin. These results provide the basis for the determination of the subsequent metabolic pathways and the identification of key proteins.
4. The transcripts of pathogenesis related proteins were differentially expressed after inoculated with S. scitamineum, involving10glucanase genes,26chitinase genes and1catalase gene. Studies on proteome associated with transcriptome showed pathogenesis-related protein1and beta-1,-3-glucanase are induced by S. scitamineum. As we know, pathogenesis related proteins are specific proteins induced by a sort of pathological conditions. They play an important role in the process of plant disease resistance reaction, and are closely related to systemic acquired resistance (SAR). According to the differential expression transcripts of these three kinds of typical pathogenesis related proteins, two β-1,3-glucanase genes ScGluAl and ScGluDl, one chitinase gene ScChi and one catalase gene ScCAT1were isolated from sugarcane by RT-PCR assay. The character of the spatio-temporal expression in differential expression genes was elucidated by Q-PCR. Subcellular location of fusion protein and the genetic transformation research of the target genes were applied by genetic engineering technology. This work will contribute to further genetic improvement of smut resistance in sugarcane by providing the excellent gene resources or technique.
(1) β-1,3-glucanase can hydrolyze the main component of fungal cell wall of β-1,3-glucan, has been shown to express during plant-pathogen interaction and involved in plant defense response. In this study, β-1,3-glucanase enzyme activity in the resistant cultivar increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1(GenBank Accession No. KC848050) and ScGluD1(GenBank Accession No. KC848051), were cloned and characterized. Predicted amino acid sequences viz both ScGluAl and ScGluDl genes contain a glycosyl hydrolases family17domain, between which only29.25%consistency at the amino acid sequence level. Phylogenetic analysis suggested that ScGluAl and ScGluDl clustered within subfamily A and subfamily D, respectively. Agrobacterium-mediated transformation for transient expression of target genes (ScGluAl and ScGluDl) with green fluorescent protein (GFP) in onion epidermal peels demonstrated that both gene products were targeted to apoplast. Escherich coli Rosetta (DE3) cells expressing ScGluA1and ScGluD1showed varying degrees of tolerance to NaCl, CdCl2, PEG, CuCl2and ZnSO4·Q-PCR analysis showed up-regulation of ScGluA1and slight down-regulation of ScGluDl in response to S. scitamineum infection. It suggested that ScGluA1may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1was inhibited. Gene expression patterns of ScGluA1and ScGluDl, in response to other factors such as salicylic acid (SA), methyl jasmonate (MeJA), abscissic acid (ABA), NaCl, CdCl2and natural drought stresses, were similar to sugarcane response against smut infection. Over expression of ScGluA1in Nicotiana benthamiana showed an antimicrobial action on Fusarium solani var. coeruleum and Botrytis cinerea. Moreover, β-1,3-glucanase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. Together, β-1,3-glucanase may work as a component in sugarcane defense mechanisms for S. scitamineum. The positive responses of ScGluAl may contribute sugarcane resistance to adversity stress; while the negative responses of ScGluDl to biotic and abiotic stresses indicate it plays a different role from that of ScGluA1in interaction between sugarcane and biotic or abiotic stresses.
(2) Chitinases (EC3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank Accession No. KF664180), a Class III chitinase gene encoded a31.37kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Q-PCR results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with S. scitamineum. The ScChi also showed obvious induction of transcription after treatment with SA, H2O2, MeJA, ABA, NaCl, CuCl2, PEG and low temperature (4℃). The expression levels of ScChi and six immunity associate marker genes were up-regulated by the transient over expression of ScChi. The histochemical assay of Nicotiana benthamiana leaves over-expressing pCAMBIA1301-ScChi exhibited deep DAB staining color and high conductivity, indicating the high level of H2O2accumulation. Besides, over-expressing ScChi helped improve N. benthamiana to defense Pseudomonas solanacearum, Fusarium solani var. coeruleum and Botrytis cinerea. Meanwhile, chitinase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.
(3) Catalase is an iron porphyrin enzyme, which serves as an efficient scavenger of reactive oxygen species (ROS) to avoid oxidative damage. In sugarcane, the enzymatic activity of catalase in the cultivar (Yacheng05-179) resistant to the smut pathogen S. scitamineum was always higher than that of the susceptible cultivar (Liucheng03-182), suggesting that catalase activity may have a positive correlation with smut resistance in sugarcane. To understand the function of catalase at the molecular level, a cDNA sequence of ScCAT1(GenBank Accession No. KF664183), was isolated from sugarcane infected by S. scitamineum. ScCAT1was predicted to encode492amino acid residues, and its deduced amino acid sequence shared a high degree of homology with other plant catalases. Enhanced growth of ScCAT1in recombinant Escherichia coli Rosetta cells under the stresses of CuCl2, CdCl2and NaCl indicated its high tolerance. Q-PCR results showed that ScCATl was expressed at relatively high levels in the bud, whereas expression was moderate in stem epidermis and stem pith. Different kinds of stresses, including S. scitamineum challenge, plant hormones (SA, MeJA and ABA) treatments, oxidative (H2O2) stress, heavy metal (CuCl2) and hyper-osmotic (PEG and NaC1) stresses, triggered a significant induction of ScCATl. The ScCAT1protein appeared to localize in plasma membrane and cytoplasm. Furthermore, histochemical assays using DAB and trypan blue staining, as well as conductivity measurement, indicated that ScCAT1may confer the sugarcane immunity. In conclusion, the positive response of ScCAT1to biotic and abiotic stresses suggests that ScCAT1is involved in protection of sugarcane against reactive oxidant-related environmental stimuli.
引文
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