【作者】 钟云；

【导师】 易干军； 邓子牛；

【作者基本信息】 湖南农业大学 ， 果树学， 2012， 博士

【摘要】 柑橘黄龙病病原为韧皮部杆菌属细菌,能侵染柑橘各种类、栽培种和杂交种,以及柑橘近缘属植物,是国内外重要植物检疫性有害生物。迄今尚未发现抗病砧木或抗病柑橘种质资源及根治柑橘黄龙病的有效方法。抗病育种应是解决柑橘黄龙病防治的根本途径,而进行柑橘黄龙病病原与柑橘相互作用机制的研究,挖掘寄主的感病机理或耐抗黄龙病病原的有效基因,无疑会为抗病育种工作奠定基础。本试验采用的材料与方法：一是选对黄龙病病原菌敏感的椪柑叶片为材料,采用抑制性差减杂交技术(SSH)研究其感染HLB病菌后早期基因变化情况；采用Solexa测序分析椪柑在发病显症期基因表达情况。旨在探索椪柑易感黄龙病病原的分子机制。二是选用广东主栽砧木资源—江西红橘的根系为材料,采用RNA-sequencing深度测序及iTRAQ技术分析其感染黄龙病病原后差异表达基因及差异表达蛋白,旨在了解江西红橘根系对病原菌的应答机制,为下一步对感病基因的抑制等研究奠定基础。以上序列库都采用实时PCR (qPCR)进行验证。主要研究结论如下：1、以实生苗‘和平椪柑’(Citrus reticulata Blanco)为材料,采用SSH技术,分别以感染黄龙病与未感染黄龙病的椪柑叶片为检测方(tester)和驱动方(driver),成功构建了黄龙病诱导的差减cDNA文库。挑选了100个阳性克隆并成功测序得到71条EST,经NCBI基因库同源性比对,有41条非冗余高质量EST序列找到了同源序列,有10条非冗余EST未搜索到同源序列。同源序列的基因涉及抗逆防御、运输、能量代谢、光合作用、蛋白代谢、信号转导、抗氧化等代谢途径和生理生化过程。值得注意的是文库中有由病原引起的韧皮部相关的凝集素蛋白前体积累。挑选了2条进行qPCR定量分析,结果表明感病1周表达量增强不大,2周后tester的表达量明显高于driver,说明材料感病早期所克隆基因表达增强。2、以SSH文库构建的同一批椪柑材料,于接种黄龙病病原后第13周、26周取样2次,分别进行Solexa测序,测序结果以clementine unigene为参考基因进行比对,并用qPCR验证,功能注释后,分别分析了各时间点的差异表达基因。结果表明：①上调差异基因比列由第13周的37%上升到第26周的64%,其中差异表达倍数>8的上调基因比列由16.7%上升到87.3%; GO process功能富集分析显示,oxidationreduction功能基因比列由第13周的4.41%上升到第26周的8.48%,其对应的response to stress的功能基因也由1.10%上升至2.08%,但defense response的功能基因由0.74%降至0.64%；相反的是,下调的defense response功能DEGs由0.55%升至0.79%。因此,从基因整体水平来看,椪柑在黄龙病原菌的侵染下,在较短时间内(3-6个月)受到的胁迫压力不断上升,抗逆表达水平得到加强,但防御能力却在下降,这可能是椪柑易感病发病的总体原因所在。②第13周及第26周的光合代谢途径的差异基因全部下调,表明黄龙病感染的椪柑植株的光合作用受到抑制。③植株与病原菌的互作途径中调控因子RIN4(RPM1interacting protein4)基因上调了8.9倍,但RPM1基因并未上调,这可能是椪柑对黄龙病菌的感病机制之一。3、以江西红橘接种黄龙病病原后第50天的根系为材料,运用RNA-sequencing进行深度测序,测序结果与clementine基因组进行比对,并用qPCR验证,功能注释后,分析了其感病后的差异表达基因,结果表明：①获得差异表达基因3956个,其中差异倍数>10的65个。显著性富集的pathways有19个,其中植物与病原互作代谢途径的显著性极高。②调控因子RIN4基因也受到上调(2.9倍),不及椪柑的上调倍数。③水杨酸信号转导途径上调明显,其中关键基因非表达子1(NPR1)上调1.6倍,下游PRl抗病基因上调2.4倍。说明红橘对黄龙病菌具有一定的系统性获得抗性。不过茉莉酸信号转导途径并未发挥作用,可能与黄龙病菌诱导相关性不大。④糖代谢途径中,蔗糖转化酶基因上调了1.7倍,而蔗糖合成酶基因下调了1.8倍,说明根系组织染病后蔗糖含量梯度变低。这可能与韧皮部堵塞、蔗糖供应减少有关。另外,铁还原氧化酶(下调2.2倍)受到了抑制,可能与根系营养不足有关。4、运用iTRAQ技术,分析了江西红橘接种黄龙病病原后第50天的根系的蛋白差异表达情况,结果表明：①鉴定到的蛋白数量为1455,其中差异性高的蛋白有78个。差异蛋白主要参与生物代谢、防御反应、抗氧化、转运和几丁质代谢等。②与转录组关联性强的差异蛋白有36个,其中半数与抗病(逆)相关,如热休克蛋白、过氧化物酶、奇异果样蛋白、几丁质酶、枯草杆菌样蛋白酶、筛管阻塞蛋白等。③植株感病后韧皮部筛管闭塞有关的筛管阻塞蛋白上调了1.67倍,这与防御黄龙病病原有关。另外,枯草杆菌样蛋白酶表达是对照的282.09倍,其相关基因表达上调了3.14倍,说明该酶参与了黄龙病原菌的抵御及根尖分生区的保护。更多还原

【Abstract】 Huanglongbing (HLB), the most destructive. serious and uncontrollable citrus disease, is caused by a phloem-restricted bacteria, named Candidatus Liberibacter, Which can infect nearly all citrus species, cultivars and hybrids, as well as some citrus relatives. HLB has been an important harmful plant quarantine disease both at home and abroad. Neither HLB resistant rootstocks and citrus germplasm resources nor effective and durable HLB control methods have so far been found. The commonly used practices in controlling HLB spread are using disease free nursery materials and using chemicals to control its vector insect citrus psyllid in addition to eradicating the infected trees. Apparently, further researches on the plant-pathogen interaction mechanisms are needed to help us in understanding the host pathogenesis and in finding possible solutions to the disease such as potential resistance genes that could be used in breeding for HLB resistance.In this study. two citrus varieties(Citrus reticulata Blanco), Ponkan, a HLB sensitive variety, and Jiangxi red tangerine, one of the main rootstock in Guangdong province and seems to be HLB tolerable, were characterized for their reactions to HLB infection at transcriptomic or proteomic levels. Ponkan asymptomatic leaves from the early stage of HLB infection and symptomatic leaves showing blotchy mottle yellowing were used for the construction of a SSH (suppression subtractive hybridization) cDNA library, and were compared for their differences in gene expression patterns by Solexa sequencing of RNAs. Root samples of Jiangxi red tangerine were subjected to RNA-sequencing for profiling the differentially expressed genes (DEGs) and to isobaric tags for relative and absolute quantification (iTRAQ) analysis for detecting differentially expressed proteins. The expression patterns of some DEGs in these experiments were further confirmed by quantitative RT-PCR (qPCR). Results are shown as follows:1、A SSH library was successfully constructed using cDNA synthesized from RNA extracted from leaves of ponkan infected with HLB as tester and uninfected as driver. One hundred positive clones were randomly selected and sequenced. and71ESTs were obtained. A search based against NCBI GenBank, after removing the repeats and low quality sequences, revealed that41ESTs share considerable homology with known genes and that10ESTs did not have significant matches. Functional annotation of the genes showed that they were related to metabolic pathways and physiological and biochemical processes such as stress-tolerance, transportation, energy metabolism, photosynthesis, proteometabolism. signaling and anti-oxidation. It is noteworthy that the lectin protein precursor gene that is commonly induced by pathogen was found in the HLB bacteria-infected Ponkan leaf cDNA library. qPCR results showed that those two up-regulated genes were HLB induced. The above results indicated that an active anti-infection reaction was initiated in Ponkan leaves during the early stage of HLB bacteria infection.2、Ponkan leaves of13weeks and26weeks after HLB inoculation were used for digital gene expression analysis. BLAST search of the clementine unigene using the sequencing results were carried out. Some of the DEGs were confirmed with qPCR. Results indicated that:①The number of up-regulated genes increased from37%in13wpi (weeks post inoculation) to64%in26wpi. what is more, the DEGs with fold change increased more than8times were increased from16.7%to87.3%. Gene ontology (GO) process molecular function enrichment analysis showed that the DEGs with oxidation reduction function increased from4.41%to8.48%and that DEGs responsive to stresses increased from1.10%to2.08%. but these related to defense responses decreased from0.74%to0.64%. However, those related to defense responses of down-regulated genes increased from0.55%to0.79%. Apparently, the expression level of resistance genes strengthened, while the defense ability of host declined along with enhanced stresses caused by HLB infection.②Photosynthesis related genes were down-regulated in both13wpi and26wpi, which indicated that HLB infection greatly reduced the citrus photosynthesis, perhaps via feedback regulation of the accumulated starches resulted from blockage of sieve tubes by the bacteria in the phloem tissue.③RIN4，a negative regulator in plant-pathogen interaction, was found up-regulated by approximately9-fold. The up-regulated expression of RIN4might play important roles in promoting bacteria growth.3、Jiangxi red tangerine root samples at50wpi were used in RNA-sequencing. Some DEGs were confirmed by qPCR. Results showed that:①3956genes were differentially expressed, among which65DEGs were identified with more than10-fold changes. DEGs were involving in19pieces of notable pathways, and the plant-pathogen interaction pathway related DEGs were enrichment very significantly.②RIN4was up-regulated about2.9times, which was much less than that in Ponkan, which may account for the difference in syndromes between the two cultivars.③SA signal transduction pathway related gene was up-regulated, the key gene of this pathway, NPR1was up-regulated1.6times, and the downstream PR1gene were up-regulated2.4times. JA signal transduction pathway related gene didn’t have notable responses to HLB infection.④Sucrose invertase was up-regulated1.7times, while the sucrose synthase were down-regulated1.8times. This may indicate that the sucrose level in HLB infected roots decreased as result of plugged phloem. Furthermore, ferredoxin expression was down-regulated2.2times, which may be related to nutrition deficiency.4、Comparative proteomic approach was also applied to decipher the pathogenic process of HLB in affected red tangerine roots using iTRAQ technique. Results showed that:①The1445identified proteins mainly ranged from20to70kDa. Differentially expressed proteins were mainly involved in metabolism, defense reaction, antioxidation, transport and chitin metabolism.②36proteins showed high correlation with transcriptome results, and half of them were stress/disease resistance related, such as heat shock proteins, resistance proteins, peroxidase, miraculin-like proteins, chitinase, subtilisin-like protease and sieve element occlusion (SEO).③plugged phloem related protein SEO was up-regulated by1.67times, and this protein may function in inhibiting infection of the HLB bacteria and hampering the long distance transport of sucrose. Subtilisin-like protease was up-regulated with a fold change of282.09and the gene of this protein was also up-regulated3.14times, which suggested that the enzyme play important roles in inhibiting the infection of HLB bacteria and protecting the root meristematic zone.更多还原