【作者】 王辉；

【导师】 王国平； 易干军；

【作者基本信息】 华中农业大学 ， 植物病理， 2011， 博士

【摘要】 柑橘黄龙病,也称作青果病,是柑橘上最严重的病害之一。黄龙病防治至今还没有有效的根治方法。柑橘木虱(Diaphorina citri)是黄龙病唯一的传播媒介。九里香(Murrava paniculata)为芸香科植物九里香属植物,是柑橘木虱最喜食的植物之一,感染黄龙病后很少表现症状。九里香作为抗黄龙病材料已有研究,但关于九里香感染黄龙病菌后基因表达情况研究很少。本研究采集田间具有典型黄龙病症状的样品,并对样品嫁接进行室内生物学鉴定、PCR扩增确定柑橘黄龙病阳性样品。在室外自然条件下研究柑橘木虱对福建茶和芸香科植物的选择性,观察柑橘木虱在福建茶和九里香上的生活周期,并在室内条件下研究柑橘木虱不同龄期若虫和成虫的饥渴耐受时间。采用不同浓度海芋乙醇提取物处理柑橘木虱若虫和成虫,采用夹毒叶片法分别测定提取物对柑橘木虱致死率,研究海芋提取物对柑橘木虱的生物活性。比较九里香感染黄龙病的不同途径,找出最佳感染途径后,收集感染黄龙病的九里香作为研究材料,应用SSH方法构建了九里香感染黄龙病早期的差减cDNA文库,分离获得在黄龙病病程相关的相关的cDNA片段,从中筛选出与抗性相关的基因片段。利用高效热不对称PCR技术扩增感染黄龙病菌的九里香的STH-21的cDNA全长,旨在克隆九里香上与黄龙病相关蛋白的STH-21蛋白的全长定量检测表达情况。本文主要研究结果如下：柑橘木虱可在福建茶和九里香上生存繁殖,两者若虫历期和成虫历期差异较大,福建茶上若虫期(15.0-20d),成虫期(17-19d),对照九里香上分别是15.0～17.0d和20.0～23.0d,福建茶上生活周期和九里香上相近,福建茶上为39.0-44.0d,九里香为39.0-45.0d九里香上若虫发育为成虫成活率为16.9%。比九里香低。福建茶对柑橘木虱是一种可接受寄主而不是最适合的寄主.柑橘木虱对寄主选择性测定显示该虫在选择各寄主之间差异明显,最适寄主为黄皮和九里香,其次是柠檬,沙田柚、红江橙和椪柑差异不大,很少取食枳壳。耐饥渴能力测试结果表明,低龄若虫和高龄若虫在供水缺食物时平均存活时间分别为10.0～12.0h和39.0～40.0 h,成虫可存活69.0-75.0h；在缺水供食物的条件下,平均存活时间分别为8.0-8.8h和35.0-37.0h,成虫为48.0-50.0h,在缺水缺食物的情况下,平均存活为7.7-8.2 h和15.0-18.0左右,成虫为36.0-38.0h。水分能延长柑橘木虱存活时间,成虫的耐受能力强于若虫。海芋挥发性物质和不同浓度乙醇提取物毒杀效果表明：挥发性气体物质0.5h内对柑橘木虱有强效毒杀作用。各级浓度提取物乙醇溶液对柑橘木虱成虫和若虫均有不同程度的杀灭效果。10克/100mL对木虱才有很强的毒杀作用,3h时每组死亡的平均数量为9.3,7h后几乎全部死亡,校正死亡率分别为45.6%和98.4%。提取物浓度为5克/100mL时,3H和24h后每组的平均校正死亡率分别为23.5%和71.0%。随着浓度的降低到0.1克/100mL,平均校正死亡率分别为8.5%和11.5%基本失去了毒杀作用。采用汁液摩擦、注射和挤压法,接种2个月后不能在九里香植株体内检测到黄龙病菌；黄龙病阳性接穗红江橙(Citrus sinensis Osbeck)嫁接九里香能使黄龙病菌成功侵染九里香,但接穗不能抽芽。草地菟丝子(Cuscuta campestris)和虫媒柑橘木虱(Diaphorina citri)都能成功从阳性植株体内将黄龙病菌传播至九里香。柑橘木虱传“毒”效率结果显示：木虱在红江橙阳性植株上取食1.5h-2h就能携带黄龙病菌,病原从柑橘木虱传到健康的九里香上最短需要12h,单头木虱带菌就能使九里香成功感染黄龙病菌。温度对病原的传播效率有较大影响,气温高于35℃或低于15℃时,介体昆虫活力差,传播病原的效率较低,0℃温度下木虱几乎不传播病原。40-C时昆虫死亡率高,活力差。采用抑制差减杂交技术,构建了柑橘黄龙病菌侵染九里香后不同时段的混合样品SSH正向和反向文库。Nest-PCR结果表明,插入片段大小平均为400 bp大小从0.2 kb到lkb不等。通过反向Northern斑点杂交技术对两个差减文库中400个片段进行筛选(正向300个,反向100个),共获得了167个差异EST表达片段。其中黄龙病菌诱导寄主上调表达的基因115个,下调表达的基因52个。利用BLASTn和BLASTx对所得EST进行相似性分析,结果显示,目前在柑橘中报道的仅有10个,其它绝大部分来源于拟南芥、蓖麻属、和番茄等。在167个ESTs片段中,36%尚不能推断明确其功能,其余64%涉及抗病防御、转录、信号转导、能量、代谢、蛋白质合成等信号途径,提示黄龙病菌与寄主互作是一个多基因参与的过程。研究结果对认识黄龙病菌-寄主互作的分子机制和寻找抗病相关基因有理论与实践意义。半定量PCR用来检测文库中miraculin-like protein, sthp2-21 ubiquitin-conjugating enzyme 8, chlorophyll A/B binding protein片段感染黄龙病菌前期表达情况,结果显示,miraculin-like protein, sthp2-21, ubiquitin-conjugating enzyme8属于上调表达基因,chlorophyll A/B binding protein属于下调表达基因,获得了九里香病程相关蛋白STH21基因的cDNA全长序列。该基因与葡萄属(fragaria)的同源性为68%,与马铃薯pSTH-21基因核酸序列的同源性为67%,与其它植物的同源性为50%～60%。半定量RT-PCR分析表明,黄龙病菌感染两周后该基因的表达量达到高峰。更多还原

【Abstract】 Citrus Huanglongbing (HLB), also called citrus greening disease is the most destructive and devastating disease of citrus in the world, No effective controls of citrus greening disease.Now. Diaphorina citri is only a insect vector tranmitted HLB. Murraya paniculata is Rutaceae, is made for host.also is host of hlb Murraya paniculata infrequently shown symptom after infected hlb, it was studied as a resistence material to HLB. there is a little paper on the status of gene express infected HLB.In this paper, some field samples were collected with typical symptom of HLB, identification of biology chacter and PCR was carried in the laboratory. Experiments under nature environment were conducted to the host preferences of Diaphorina citri to Carmona microphylla andvarious hosts of Rutaceae. The life cycle on the C. microphylla and Murraya paniculata were recorded and analyzed under the natural conditions. The tolerance to starvation and thirstiness of different aged nymphs and adult D. citri were tested in the laboratory condition with the situation in lack of food and water. Different concent rations of ethanol extract of Alocasia macrorrhizos leaves.were used to deal with larvae and adults of Diaphorina citr..i. Folders micro-poisonous leaves method were measured.Different transmission methods that Candidatus Liberibacter asiaticus infected Murraya paniculata, were compared and Nested-PCR was used to confirm the presence of Ca. Liberibacter asiaticus two months after inoculation. Complex cDNA library of Murraya paniculata with infected Citrus HuangLongBing at various times was constructed using suppression subtractive hybridization (SSH). according to EST from SSH library On Murraya paniculats pathogenesis-related protein STH-21 gene (accession numberU21849), using hiTAIL-PCR technology, a full-length cDNA of pathogenesis-related protein named sth- 21 was obtained from Murraya paniculats. A semi RT-PCR system was established to detect the gene expression of miraculin-like protein, sthp2-21, ubiquitin-conjugating enzyme 8, chlorophyll A/B binding protein in Murraya paniculata with HLB in the earlier stages.1 The results shows Diaphorina citri can survive on Carmona microphylla, With development duration of one generation Diaphorina citri fed on Carmona microphylla hosts varied significantly, with the long nymphaes ages(15-20d) and the adult nymphaes ages(17-19d).as contrast host., Murraya paniculata,15.0～17.0d and 20.0～23.0d respectively., and the life cycle in Carmona microphylla was close to Murraya paniculata, while the surviving rate is lower.about 16.9%. Carmona microphylla is an actetable host, rather than an optimum host. The results showed that the tendency fed on different hosts varied significantly, Murraya paniculata and clausena were favorite host for Diaphorina citri, Citrus. limon is inferior to the former, There were no significant differences between Citrus sinensis Osbeck and C. grandis (L) Osbeck and Citrus reticulata Blanco cv. Ponkan. Diaphorina citri seldom feed on Poncirus trifoliata (L) Raf.. The starvation and thirstiness tolerance of adults was described as the mean survival time. Thev results showed that in the three treatments as mentioned, the mean survival time of young and old nymphae age and adults were7.7～8.2h,15.0h～18.0h,36.0h～38.0h respectively when no food and water..10.0h～12.0h and35.0h～37.0h and 48.0h～50.0h respectively when providing food without water.11.0h～13.0h and 39.0h～40.0h and 69.0h～75h.0h when providing water without food. Water would prolong the mean survival time of adults significantly, starvation tolerance and thirstiness tolerance of adults adults were stronger than nymphae ages. Results indicated that all levels of the concent rations of ethanol extract have different levels of killing effect. The corrected mortality were 45.6%,98.4%,23.5%,71.0%,3.89%,8.5% and 11.5% by the 3 concentrations of Alocasia macrorrhizos treated with 10g/100mL,1g/100mL,0.1 g/100mL extract of ethanol, Volatile ingredients of Alocasia macrorrhizo has strongly killing effect at 0.5h。2 The results indicated that mechanical inoculation, injection and squeezing methods failed to transmit Ca. Liberibacter asiaticus to Murraya paniculata, whereas grafting from infected Hongjiang Sweet Orange (Citrus sinensis Osbeck) was successful in spite of no scions elongation, and both dodder (Cuscuta campestris) and psyllid (Diaphorina citri) were able to successfully transmit HLB bacterium from citrus to Murraya paniculata. One psyllid was able to transmit it successfully with the shortest time of psyllid feeding on Hongjiang Sweet Orange,1.5h to 2h, and 12h for Diaphorina citri to transmit HLB bacterium to Murraya paniculata, respectively. Efficient temperatures for HLB transmission by psyllid were between 15℃and 35℃, and there were no transmission of HLB bacterium below 0℃and over 40℃. The study laid a foundation for the large scale screening of resistant-related gene to Ca. Liberibacter asiaticus from Murraya paniculata.3 Two differentially expressed genes libraries-a library of up-regulated genes and a library of down-regulated genes in HLB-infected Mexican lime (C. aurantifolia) of in-vitro cultures were prepared by suppression subtractive hybridization (SSH).400 clones were isolated from cDNA library, nested PCR results show that the insert sizes ranged from 0.2kb to lkb,300 randomly selected colonies from the forward-subtractive library and of 100 colonies from the reverse-subtractive library were identified with the reverse Northern dot-blot screening method. All ESTs similarity were analysed by BLASTn and BLASTx. Sequencing results showed that167 ESTs represented different gene fragments,among which 115 genes are up-regulated expression and 52 genes are down-regulated expression by HLB infection.Among the 167 ESTs isolated and identified in Murraya paniculata. only 10ESTs had been reported on citrus in GenBank database, the others are mostly from Arabidopsis, Solanum and Ricinus communis. The function of 63 (about 36%) gene fragments was clearly unknown, while the rest 64% had function clarifying as disease defense, transcription,signal transduction, energy, metabolism and protein synthesis.The results indicated that the process of HLB-host interactions was complex and many genes could participate in the process. In this paper, we first reported the differentially expressed genes of Murraya paniculata induced byHLB infection, which established the foundation for understanding the molecular mechanisms and found disease-resistant genes.4 The RT-PCR results showed that the gene of miraculin-like protein, sthp2-21, ubiquitin-conjugating enzyme 8 were up regulated and chlorophyll A/B binding protein down regulated. Sequence analysis indicated that clonedSTH-21 gene consisted of 361 nucleotides (nt) containing 74 amino acids. Further comparison toSTH- 21 gene and Solanum tuberosum, Vitis vinifer showed that its identitywas 68% and 67%, respectively. Semi-quantitative RT-PCR revealed that expression of sth- 21 gene was induced by HLB, and the most abundantly time更多还原