【作者】 缪卫国；

【导师】 王金生；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 棉花是世界上重要的纤维作物,也是重要的油料来源。在许多植棉国家,棉花黄萎病、枯萎病是危害棉花的两大病害。从1993年以来,黄萎病在我国严重发生,发病面积占全国植棉面积的50%左右,每年损失皮棉约10万吨。目前尚无有效的方法控制该类病害的危害。通过植物基因工程,可以把优良抗性基因转移到植物体内,对植物进行定向改造,从而培育新的优良抗病品种,为棉花黄、枯萎病的有效防治开辟了新的防治途径。已经证明harpin是一类蛋白激发子,具有无毒、无公害和对环境友好等优点。先前研究表明喷施harpin能增强植物抗病、抗虫性,而内源表达harpin显示转基因植物(水稻、烟草、梨等)能抗多种病害。由此,通过基因工程方法将harpin编码基因hrp导入重要的经济作物棉花中,是否会使棉花获得各种抗性及其它有益表型呢?此外,harpin在分子水平上的作用机理和作用位点还不是很清楚,尤其在无胁迫下内源表达的harpin对植物的防卫、生长发育会产生怎样的影响?本研究将来自水稻白叶枯病菌的hpa1xoo基因转入棉花,使harpin的多种功能在棉花中得以表达,获得抗棉花黄、枯萎病等多种有益表型的转基因棉花株系,并力图解析harpin对植物的作用机理；与此同时,我们还从棉花角斑病菌(X. citri subsp. malvacearum)基因组中克隆了编码harpin的hpaXm基因。现将结果分述如下：利用含有农杆菌T-DNA区的重组质粒,结合花粉管通道技术,将hpa1xoo基因分别转化陆地棉品种中棉35(Z35)、海岛棉品种新海21等棉花品种,通过苗期的卡那霉素或除草剂筛选并结合室内和病圃枯萎病和黄萎病抗性单株筛选,获得了30份转基因材料。在卡那抗性植株筛选基础上,大部分转基因植株符合孟德尔遗传规律,即后代存在3：1或者1：3的分离。部分转基因株系到T6代就具有100%的纯合株系(如T-34部分单株株系后代)。同时采用农杆菌茎尖介导法,也获得携带有hpa1xoo基因的转基因植株。选择部分转基因植株进行加代选育,在棉花黄、枯萎病病圃进行了抗病性、农艺性状评价,以及采用生测法评价转基因植株的抗虫性。供试转基因材料中,针对棉花黄萎病,获得了抗病材料(系)8个,耐病材料(系)8个,病指范围在13.75%-38.49%,转基因材料病指均低于出发品种(Z35),Z35病指为45.34%。与Z35相比,转基因棉黄萎病平均发病率减少42.9%,病指减少7.35%-26.22%；枯萎病病指减少52.46%-59.02%；立枯病病指减少58.70%-59.24%。在抗虫性调查的初步结果中,部分转基因材料(如T-3、T-34)对棉铃虫表现出较好的抗性。饲喂T-34棉叶的棉铃虫发育明显迟缓,直至死亡,5天后棉铃虫死亡率为90.95%,而饲喂Z35棉叶的棉铃虫死亡率为12.20%。这些结果表明harpinxoo的表达赋予转hpa1xoo基因棉花抗病虫性。转基因抗病材料的棉铃有大、有小。生长前期,转基因植株的株高一般低于出发品种,但开花以后至成熟,转基因材料的生长逐渐与出发品种接近,与出发品种无显著性差异。转基因棉花生育期为120-121天,长于Z35(116天)。部分转基因材料在农艺性状方面优于Z35,如转基因株系T-33,在棉花吐絮期(2007),与Z35相比,无效枝数低了0.12个,有效桃数多了0.1个；转基因株系T-34果枝数比对照Z35多了0.1个。部分转基因材料田间吐絮较畅,纤维品质有所改善。2006年通过农业部棉花纤维检测中心检测,转基因材料T-33的纤维上半部平均长度为29.0mm,对照Z35的纤维长度为28.8mm；纤维整齐度方面,所有转基因材料(85%)都高于出发品种(83%)；马克隆值方面,所有的转基因材料为B级,对照Z35为C级。转基因株系T-34纤维伸长率为5.8%,而对照(Z35)的纤维伸长率为5.5%。因此,综合诸多性状,我们选择转基因植株T-34(或T-33)作为本研究的主要对象。从PCR扩增到目的基因(hpalxoo)的阳性植株中选择了T-34和T-33,作进一步的分子鉴定。PCR Southern blot和基因组Southern blot确定了hpalxoo已经整合到棉花基因组中,且具有至少3个拷贝,提取PCR检测均为阳性的植株的可溶性蛋白,进行了Western blot分析,在PCR阳性植株中可检测到harpinxoo蛋白的表达。RT-PCR结果同样证实了hpa1xoo在转录水平的表达。采用免疫胶体金定位技术,观察到harpinxoo免疫金颗粒主要分布在植物的细胞壁上,胶体金颗粒在转基因植物细胞壁上呈10-20粒簇状特异性分布,在叶绿体或线粒体上则未见到特异性吸附,这个结果说明harpinxoo在转基因棉花中作用位点是细胞壁。同时,采用PCR和Bt蛋白检测试纸条方法检测转基因材料中是否含有Bt基因及其编码蛋白,结果表明供检测的转hpa1xoo基因棉花对棉铃虫的抗性是由于取食表达harpinxoo 6勺植株引起的,而与Bt基因无关。表达harpinxoo转基因棉花材料T-34在田间和室内均对棉花黄萎病表现出较好的抗性,因此,我们进一步从细胞和分子水平上验证T-34对棉花黄萎病菌的抗性机理。通过T-34悬浮细胞与棉花黄萎病菌分生孢子共培养,转基因棉花T-34的悬浮细胞的生存能力显著高于其出发品种Z35。在无病原菌侵染时,T-34叶片的H202积累高于Z35,但未观察到肉眼可见的氧爆发(reactive oxygen intermediates, ROI)现象,说明T-34较Z35更易处于一种引发状态(Priming state),一旦遭到病原菌侵染,T-34叶片中的过氧化氢酶受到抑制,在T-34叶组织中可以观察到明显的氧爆发和微过敏(Micro HR)现象,H202含量也有较高的积累,并呈现病原菌与植物非亲和互作的双峰典型特征,而在Z35叶组织中没有观察到氧爆发和微过敏。与这种现象一致,通过Real Time-PCR检测,氧爆发和微过敏的标志基因ghAOXl、hsr203J及hinl等基因的相对表达量也显著高于出发品种。这些结果证明了,由于harpinxoo在转基因植物体中的表达,激活了植株对病原菌攻击的防卫反应,harpinxoo是植物与病原菌非亲合互作中必要的激发子。Harpin赋予转基因棉花多种抗性和多个有益的农艺性状表型,但是在无胁迫下harpin影响植株的分子模式还不清楚。为此我们使用棉花12K cDNA芯片,采用生物芯片技术,分析了harpinxoo转基因棉花(T-34)与其出发品种(Z35)的全基因组转录谱,从而使我们能够广泛理解harpinxoo是如何在转基因棉花中调控基因表达的。结果显示,与Z35相比,在T-34叶片中存在530个差异表达基因。这些差异表达基因中很多基因涉及过敏性细胞死亡(hypersensitive cell death, HCD)、苯丙氨酸解氨酶(phenylalanine ammonia lyase, PAL)、水杨酸(salicylic acid, SA)、茉莉酸(jasmonic acid,JA)、生长素(auxin)、脱落酸(abscisic acid)和乙烯(ethylene, ET)等基本防卫反应途径。在转基因棉花中,Harpinxoo作为内源激发子,与受体作用或者直接作用于一些蛋白激酶,造成编码NADPH氧化酶、NAD泛素氧化还原酶、离子通道蛋白(如CLC-C)钙结合蛋白、苯丙氨酸裂解酶(PAL2)、植保素合成酶(如CHS)、MAPK激酶等蛋白酶的基因上调表达,并通过乙烯或生长素转录因子(如ERF、ARF)等进一步激活下游防卫和生长发育有关的反应。Harpinxoo在转基因棉花中调控生长素相关基因的表达符合生长素信号途径；同时,在LRR-PRKs途径中多个激酶编码基因的表达,表明LRR-PRKs在harpinxoo诱导的信号传导中也担任重要角色。通过对芯片上9个防卫相关基因采用定量或半定量PCR验证,显示芯片数据具有88.98%的可信度。经Realtime PCR检测,病原菌侵染后,6个防卫相关基因在转基因植株中超表达。这个结果进一步说明在无胁迫下harpinxoo的表达赋予转基因棉防卫水平高于其受体,并保持着基本的防卫水平,但在遭受病原菌胁迫下,harpinxoo的表达具有增强转hpalxoo基因棉防卫水平的能力。同时,差异表达基因中的共性上调和下调表达以及涉及能量产生和消耗途径的基因表达水平的提高,这些结果意味着在无胁迫下转基因植物体中维持着一种能量平衡,仅仅当病原菌侵染时才在植物体中产生高能量的需求。用转hpa1xoo基因棉花新鲜叶片饲喂棉铃虫,造成棉铃虫发育明显迟缓。为了更好地解析这一现象,将转基因棉花T-34和出发品种Z35叶片分别饲喂棉铃虫,提取棉铃虫幼虫RNA,利用和棉铃虫同属鳞翅目的家蚕的23K寡核苷酸(Oligo)探针,通过生物芯片技术,制作了其Oligo表达谱芯片。芯片结果显示检测到了2927个基因,T-34对Z35在转录水平上存在有872个差异表达基因,其中Ratio值大于2.0的上调表达基因有328个,Ratio值小于0.5的下调表达基因有544个。872个差异表达基因主要涉及13种生物功能,96个通路(pathway)。棉铃虫取食表达Harpinxoo的棉花叶片后,激发棉铃虫体内编码蛋白水解酶、细胞色素P450等的基因超表达,打破了离子调节体系和渗透压平衡体系,消耗棉铃虫的大量ATP,从而影响棉铃虫蛋白酶分解和合成、ATP合成等多个生物途径的正常代谢,造成棉铃虫不能很好取食。因此,采用芯片技术,通过异源家蚕oligo芯片信息的比对,初步解析了转hpalxoo棉花对棉铃虫的抗性机理,可能与棉铃虫取食后正常代谢受到干扰,影响发育有关。本研究结果为hpa1xoo,作为新的基因资源用于抗棉铃虫育种提供了初步证据,并为抗棉铃虫机理研究展示了新的途径。Xanthomonas campestris pv. malvacearum (E.F. Smith) Dye (Xcm)是引起棉花角斑病的病原。2006年该病菌学名更名为X. citri subsp. malvacearum nov. comb. nov. nom. nov. (Xcm)。在Xanthomonas中一些编码harpin的基因已经被克隆,但在Xcm中尚未有该基因克隆的报道。本研究首次报道了编码harpin like protein的hpaXm基因。用PCR技术从Xcm基因组中扩增了hpaXm基因。该基因具有402bp,其编码的蛋白为HpaXm,含有133个氨基酸,大小为13.3kD,GC含量为60.70%,富含甘氨酸,含量为21.80%,缺乏半胱氨酸。HpaXm的GST融合蛋白表达的最佳条件是IPTG0.05mM、37℃3h。纯化的HpaXm具有热稳定性,煮沸或未煮沸处理的HpaXm都能在烟草上激发HR.HpaXm能够增强烟草对TMV的抗病性,也能激发nprl、hsr203Jpr-la、pr-1b等防卫基因的表达。在Xcm基因组中,采用TAIL-PCR (thermal asymmetric interlaced PCR)方法,在hpaXm左翼扩增到了与X. oryzae pv. oryzae中hrcC基因具有93%同源性的1826bp序列,命名为hrcCXm,但在hrcCXm与hpaXm之间有704bp的间隔序列；在hpaXm右翼,扩增得到了与X. oryzae pv. oryzae中hpa2基因具有89%同源性的630bp序列,命名为助a2Xm。在HpaXm蛋白序列中存在有两个Alpha helix区域。HpaXm可能拥有Coiled coil区域及亮氨酸拉链。通过完整地氨基酸序列及N端a-helix中13个保守的残基(H2N-SEKQLDQLLTQLI-COOH)进化树分析,HpaXm与HpaGXac、HpaXac进化关系要比与Hpa1Xoo和Hpa1Xoc更近。我们对HpaXm N端α-helix区域中含有两个heptad的多肽(39-LDQLLTQ-LIMALLQ-52)进行合成,生物活性测定表明其能在烟草上诱导HR；改变相应残基的该多肽衍生物HpaXmAT44C和HpaXmAM48Q,显示出HR活性减弱的表型。通过信号肽软件预测和GST检测实验,在HpaXm的N端可能具有潜在的信号肽,可能以sec-dependent pathway必出胞外。更多还原

【Abstract】 The soilborne fungal pathogen, Verticillium dahliae Kleb., is the causal agent of Verticillium wilt in a wide range of crops including cotton. This fungal pathogen is commonly present in many cotton-growing areas and has been considered as a major threat to the production of cotton worldwide. The traditional breeding for cotton varieties resistant to Verticillium wilt has not yet succeeded due to the lack of genetic resources for the resistance capable of providing high level of protection against Verticillium wilt. The genetic engineering utilizing plant defense-related genes has been considered to be an alternative to generate crops with improved resistance against plant pathogens, insects or herbicides. HarpinXoo encoded by hpalXoo in hrp cluster from Xanthomonas oryzae pv. oryzae is capable of inducing defense responses including hypersensitive reactions (HR) in non-host plants.By using recombination plasmid containing T-DNA region (ligated with hpalXoo) from Agrobacterium, and companying with the pollen-tube pathway transformation method, the 30 transgenic harpinXoo plants were obtained, containing a selectable ’marker’ gene, neomycin phosphotransferaseⅡ(nptⅡ), or bar gene respectively.The genes confers resistance to the antibiotics kanamycin or herbicide glufosinate-ammonium, allowing selection of the transgenic plants from untransformed plants during growth in tissue culture and field. In the meantime, the transgenic hpalxoo plant were generated using the stem apexes as explants by Agrobacterium-mediated transformation method. By resistance screening in Verticillium wilt nursery, it were obtained for some resistant transgenic cotton plants to Verticillium dahliae, including to 8 resistant plants,8 tolerance plants, the idex of disease was arranged from 13.75% to 38.49%. The transgenic cotton plants proved to be highly resistant to cotton Verticillium wilt, average disease index of the plants to the disease reduced 7.35%-26.22%, compared with untransformed plant Z35. In addition, the transgenic plants also were obtained the higher resistance to Fusarium oxysporum f.sp. vansifectum, Rhizoctonia solani, and cotton bollworm (Helicoverpa armigera). The disease index of the plants to the two pathogens were reduced 52.46%-59.02%and 58.70%-59.24%respectively. The boll size of transgenic cotton was smaller than that of its receipt variety Z35, its height also was shorter than Z35 in the front development stage. But the whole development stages of transgenic plants were longer than that of untransformed cotton Z35. The result also showed that the expression of harpinxoo in transgenic plants promoted the fibre quality.The fresh and unwounded leaves from independent plants at the 6-7-leaf stage were collected and used for feeding 25 Helicoverpa armigera larvae. After 5 days, we investigated the larval development and the leaf damage. The T-34 and GK22 lines showed higher resistance to H. armigera; the corrected mortality of H. armigera larvae fed on T-34 leaves (LFT-34) was approximately 90.95%higher than that of the larvae fed on leaves of the untransformed cotton line Z35 (LFZ35). The development of LFT-34 slowed down, eventually leading to larval death. The 340-bp-long Cry1Ac was amplified only in the GK22 genome, while the 420-bp-long hpalxoo was amplified only in the T-34 genome, and both hpalxoo and CrylAc were not amplified in the Z35 genome. These results indicated that the resistance of the hpalxoo-expressing cotton line T-34 to H. armigera could be attributed to the expression of harpinxoo in the T-34 plants instead of the Bt proteins encoded by CrylAc.Three positive bands were obtained in each T-34 and T-33 sample, located at about 4kb, 6kb and 10kb in Southern blot pattern. The pattern suggests that T-34 incorporates the hpalxoo transgene at different chromosomal locations. Western blot analysis showed the harpinxoo protein to be constitutively expressed in the transgenic lines, whereas it was barely detectable in Z35. The molecular analysis of the transgenic cotton plants demonstrated that the coding sequence of harpinxoo was present in the recipient genome, the transgenic cotton plants had at least three copies of the hpalxoo transgene. By using Immuno-gold localization method, The special distribution of harpinxoo with gold labeled immunosorbent was only observed in the walls of leave and stem apexes cells in transgenic cotton T-33 and T-34 at 5-6 leaves stage. Although we also found gold particles in cell membrane and Chloroplast, only a few gold particles were observed in it, and it should belong to non special distribution. No any gold particles was observed in the cell walls of untransformed cotton Z35. we were not able to find any gold particles in the mitochondrial of T-34, including leave and stem apexes tissues in T-33 and Z35 plants. The distribution pattern of immunogold labeling for harpinxoo in leaves or stem apexes tissue from T-33 and T-34, was different from that for other protein in plant tissue, it mostly showed us 10-20 gold particles gathered in the cell walls of T-34 leave and stem apexes tissues. It was easy for us to observe 10-20 gold particles gathered among three cell walls in stem apexes of T-34 compared with one or two cell walls in leave or stem apexes of T-34.Hpalxoo expressed and located at in transgenic cotton cell wall, as a cell wall endogenous elicitor, triggers the generation of H2O2 in plant, H2O2 content in T-34 keeps the higher level than that in the receiptor variety Z35, T-34 plant is a priming state for avoiding the putative pathogen attack. Although the higher accumulation of H2O2 in transgenic hpalxoo plant, it is not enough to trigger the generation of reactive oxygen intermediates (ROI) in plant without pathogen attack, the expression of ROI marker gene ghAOXl in T-34 is slightly induced compared with Z35. After pathogen attack, H2O2 in T-34 is rapidly accumulated, and DAB dyeing indicates reddish spot in the plant tissue and ROI production, and thus micro HR also is induced. Two peak values of H2O2 content at 0-8h were found in T-34 rather than that in Z35, which is the character of incompatible interaction between pathogen and plant. These evidences suggest ROI and HR are directly related to the interaction of harpinxoo and pathogen. Whereas ROI and HR in transgenic hpalxoo tobacco are not found. In addition, different from transgenic hpalxoo tobacco, the expressions of hinl and hsr203J are induced in transgenic hpalxoo cotton T-34. This results are consistent with transgenic hpaGEP tobacco. However, the expression of nprl in T-34 is only slightly highter than that in Z35, nprl is induced in transgenic hpalxoo tobacco, but not in transgenic hpaGEp tobacco. The difference between transgenic hpalxoo cotton and tobacco may be caused by the different transformed receiptor of hpalxoo or the different insert sites of hpalxoo in plant genome. These evidences suggested that the successful transformation of hpalxoo into Z35 resulted in an altered regulation of genes involved not only in disease resistance but also in several other metabolic pathways.Harpinxoo, like other harpins, is able to elicit disease and insect resistance in plants, induce many plant-reaction phenotypes, and promote plant growth in yield and quality. However, the mode of action of harpin at the molecular level is not well understood. By analysis of globe transcriptome profile of transgenic harpinxoo cotton T-34 vs untransformed cotton Z35(Gossypium hirsutum L), using the customized 12K cDNA cotton microarrays, we were able to understand broadly how harpinXoo regulated the globe transcription expression of genes in transgenic harpinxoo plants. Totally,530 genes in leaves were deregulated in transgenic cotton compared with that in untransformed one. The differentially expressed genes in harpinxoo-transgenic cotton was significantly enriched in programmed cell death (PCD), phenylalanine ammonia lyase (PAL), salicylic acid (SA), jasmonic acid (JA), Auxin, abscisic acid (ABA) and ethylene (ET) pathways, as basic defense responses. Harpinxoo induced the expression of ERF related to ethylene or ARF related to auxin. Harpinxoo not only activated the genes involved in LRR-PRKs pathway which played an important role in development and defense pathways but also used MAPK pathway kinases to activate downsteam defense responses. By validating the microarray data,8 of 9 data points were consistent with the Real Time-PCR assay. In the meantime, the expression of 6 genes, which were related to defense response, was induced to be over-upregulated after challenged by Verticillium dahliae conidia. These evidences suggested two different defense pathways or abilities were adopted in T-34 while pathogen attack or not. In addition, Up-and down-regulation simultaneously in differential expressed genes and increased expression of both genes encoding energy producing and consuming pathways suggested the energy balance was maintain in the genetic modified cotton without biotic stress. High-energy demand would only occur when infected with pathogen.The silkworm genome-wide microarray was used to compare the global transcriptomic profile of the H. armigera larvae fed on T-34 leaves (LFT-34) with that of the larvae fed on leaves of the untransformed cotton line Z35 (LFZ35). The microarray data indicated that 872 genes in LFT-34 were totally deregulated, in comparison with their expression in LFZ35. Among these 872 genes,328 genes were upregulated (LFT-34/LFZ35 ratio,>2.0), and 544 genes were downregulated (LFT-34/LFZ35 ratio,<0.5). All the differentially expressed genes can be classified and related to 13 biological functions and were involved in 96 biological pathways. Harpinxoo confers T-34 with resistance to H. armigera and influences multiple metabolic pathways in H. armigera larvae. These results also showed that the silkworm microarray can be used as a new approach to analyze the genome-wide transcriptional expression of H. armigera and the interaction between H. armigera and plants or other organisms.Xanthomonas campestris pv. malvacearum (E.F. Smith) Dye (Xcm) is a bacteria blight pathogen. It causes cotton bacteria blight. In 2006, X. campestris pv. malvacearum (E.F. Smith) was renamed X. citri subsp. malvacearum nov. comb. nov. nom. nov. (Xcm, Schaad et al.2006). A novel harpin-like protein, HpaXm, was described from Xcm. Like harpins from other plant bacteria, HpaXm encoded by the hrp gene is a glycine-rich, cysteine lacking, heat-stable protein with the ability to induce the hypersensitive reaction and the enhanced expression of defense-related genes in tobacco leaves. hpaXm was found to be localized between hrp2 and hrcC by TAIL-PCR (thermal asymmetric interlaced PCR) technology. A phylogenetic analysis of the complete amino acid sequence or solely the 13 highly conserved residues H2N-SEKQLDQLLTQLI-COOH in the N-terminal a-helix indicate that HpaXm is evolutionarily closer to HpaGXag and HpaXac than to HpalXoo and HpalXoc. The synthesized peptide containing two heptads,39-LDQLLTQ-LIMALLQ-52, from the N-terminal a-helical region of HpaXm displayed a comparable activity in inducing HR, but other two synthesized derivatives, HpaXmAT44C and HpaXmAM48Q showed a reduced activity of HR. A signal peptide was predicted to exist in the 15 leading amino acids of the N-terminus. The data from a GST trap test suggests that the signal peptide may function to release HpaXm into the extracellular medium and the sec-dependent pathway may associate with HpaXm during secretion.更多还原