【作者】 王暄；

【导师】 徐建华； 李红梅；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 博士

【摘要】 植物寄生线虫是世界范围内对农业生产造成极大破坏的病原生物,其中主要的三类固定内寄生线虫,即球形胞囊属、异皮属和根结属线虫,每年给世界农业生产造成的损失约为一千亿美元,特别是南方根结线虫,由于其地理分布和寄主范围非常广泛,因而被认为是最具有破坏性的病原线虫。根结线虫侵染性二龄幼虫从靠近植物根尖的部位侵入寄主,移动至根尖顶端分生区后向上折返进入维管束,继续移动一段距离后选择5-7个植物细胞,利用口针分泌的蛋白诱导和调节植物细胞形成永久性的取食位点“巨细胞”,线虫经三次蜕皮后发育为成熟雌虫。来自根结线虫三个食道腺的分泌物被认为在线虫寄生过程中具有重要作用,因此鉴定编码线虫食道腺分泌蛋白的寄生相关基因,是明确线虫寄生寄主植物机理的基础。通过运用多种分子生物学手段,已成功克隆了多个线虫寄生相关基因,包括纤维素酶、果胶裂解酶、扩展蛋白和分支酸变位酶等,然而植物寄生线虫专性寄生以及难以转化和突变的特点,极大的阻碍了对线虫寄生基因功能的研究。RNA干涉技术的出现,为植物寄生线虫基因功能研究提供了一种有效工具,并为运用转基因技术防治根结线虫提供了新的策略。本研究运用代表性差异显示技术(RDA)分离南方根结线虫寄生相关基因,选择分离得到的推测编码的氨基酸与毒液过敏原相似的cDNA片段,克隆其全长并进行分析,并且运用活体外(in vitro)和活体内(in vivo)两种RNA干涉方式对该基因进行功能分析。以南方根结线虫寄生早期二龄幼虫和寄生期幼虫为材料,用RDA分离两个样本间的差异表达基因,在材料中同时加入了远源序列绿色荧光蛋白基因(gfp)的片段作为内标,最终筛选获得了15个cDNA片段。RT-PCR验证表明有5个片段差异表达明显,其中两个片段在寄生早期二龄幼虫特异性表达,两个片段在寄生早期二龄幼虫中高水平表达,另有一个片段在寄生期幼虫中上调表达,其余片段在两个样本间表达水平无明显差异。BLASTX分析表明,15个cDNA片段中有5个在数据库中没有同源序列,其余片段分别与已报道的植物寄生线虫、新秀丽小杆线虫及动物寄生线虫的蛋白质序列相似。选择正向RDA分离的差异表达片段Blb,根据其cDNA序列设计引物,结合快速扩增cDNA末端法(RACE)及基因组DNA扩增,获得了南方根结线虫类毒液过敏原基因(Mi-vap-2)全序列。Mi-vap-2全长1917bp,包含3个长度从29bp到797bp的内含子,以及4个37bp到361bp的外显子。Mi-vap-2的cDNA序列编码294个氨基酸的开放阅读框(ORF),包含一个预测的16个氨基酸的分泌信号肽。Southern blot分析表明该基因在南方根结线虫中可能以多拷贝或多基因家族的方式存在。原位杂交显示Mi-vap-2特异性的在南方根结线虫亚腹食道腺表达。RT-PCR分析该基因发育表达模式证实,Mi-vap-2仅在侵染性二龄幼虫和寄生早期幼虫中表达,推测该基因可能在南方根结线虫与寄主互作过程中具有重要作用。比较分析了神经刺激类物质章鱼胺(octopamine)和间苯二酚(resorcinol)在体外条件下对根结线虫二龄幼虫吞咽的刺激效果,结果显示章鱼胺对刺激根结线虫在体外条件下吞咽没有明显作用,而间苯二酚则能有效刺激二龄幼虫的吞咽；并对间苯二酚在体外条件下对刺激根结线虫摄食的方式和条件进行了优化。以^Mi-vap-2cDNA为模板,体外转录合成dsRNA,结合优化的刺激条件,对南方根结线虫类毒液过敏原基因Mi-vap-2进行了活体外(in vitro) RNA干涉诱导基因沉默的研究。RT-PCR检测显示,dsRNA的浸泡处理引起了南方根结线虫侵染性二龄幼虫体内Mi-vap-2转录水平的下降,接种实验证实处理后的线虫在感病番茄上的繁殖力降低,表明活体外RNA干涉Mi-vap-2在南方根结线虫中的表达取得了成功,并且表明Mi-vap-2对南方根结线虫寄生寄主植物可能有重要作用。通过将南方根结线虫类毒液过敏原基因Mi-vap-2的部分cDNA序列分别以正向和反向的方式插入到载体pHANNIBAL,再将含有反向重复序列的该转化单元亚克隆至二元表达载体pART27,获得了以Mi-vap-2为沉默靶标的dsRNA表达载体。在农杆菌的介导下用叶碟法将该表达载体转化烟草,通过对筛选到的T2代转基因烟草Southern blot分析,表明目标转化单元以单拷贝的方式整合到了烟草的基因组中并得到了稳定的遗传,同时通过Northern blot,在转基因烟草中检测到了与Mi-vap-2同源的小干扰RNA (siRNA)。接种实验中该转基因烟草没有引起南方根结线虫寄生能力的明显下降,表明Mi-vap-2可能不是较佳的沉默靶标来进行转基因防治根结线虫。实验结果还证实了利用寄主植物产生并递送dsRNA至线虫的RNA干涉策略是可行的。更多还原

【Abstract】 Plant parasitic nematodes are economically important pests for many crops worldwide. Three groups of endoparasitic sedentary nematodes which belong to genera Globodera, Heterodera (cyst nematodes) and Meloidogyne (root-knot nematodes, RKN), cause enormous yield losses to world agriculture estimated at US$100billion annually. Among them, the root knot nematode Meloidogyne incognita, is considered to be the most dangerous pest because of its wide geographic distribution and host range. The second stage juveniles of root knot nematode enter the plant closely behind the root tip and migrate intercellularly. Subsequently they go to the vascular cylinder where they induce a permanent feeding site, giant-cell. The juveniles moult three times to adult female. Stylet secretions originated from esophageal gland cells of root knot nematodes are thought to play an important role in the interaction of these parasites with their host plants, therefore the cloning and identification of parasitism related genes encoding esophageal gland proteins secreted from the nematode stylet into host tissues is the key to understanding the molecular basis on parasitism of plants.Some pathogenicity related genes including cellulase, pectate lyase, expansin and chorismate mutase have been successfully cloned from some nematodes species using different molecular techniques. Nevertheless, the obligate biotrophic nature and refractory to mutation and transformation allow the functional analysis of genes more difficult. The development of RNA interference (RNAi) technology provides an effective tool for understanding gene function in plant parasitic nematodes and a new transgenetic strategy for controlling root-knot nematodes.In this study, the parasitism related genes from M. incognita were isolated by representational difference analysis (RDA); one isolated cDNA fragment which deduced amino acid sequences similar to venom allergen-like protein, its full length cDNA was cloned by rapid amplification of cDNA ends (RACE) and analyzed;, in vitro and in vivo RNAi research was carried out to validate the function of the isolated gene in parasitism of M. incognita. A representational difference analysis (RDA)-based strategy was used to identify parasitism related genes differentially expressed in two different parasitic stages of root knot nematode M. incognita, early parasitic second-stage juveniles and parasitic-stage juveniles. The DNA fragments derived from green fluorescent protein(gfp) were used as internal standards to monitor the effect of subtractive hybridization. Total of15cDNA fragments were screened and identified. Five out of15cDNA fragments were proved to be differentially expressed by reverse transcription-polymerase chain reaction (RT-PCR). Two cDNA fragments were found to be presented in the early parasitic second-stage juveniles and absented in the parasitic-stage juveniles, two were up-regulating expressed in the early parasitic second-stage juveniles and one was up-regulating expressed in parasitic-stage juveniles. In BLASTX searching and analysis, the sequences of5cDNA fragments did not show any significant similarity with sequences from known proteins, while the rest of ten were highly matched with sequences of reported proteins from phytoparasitic nematodes, free-living nematode Caenorhabditis elegans and animal parasitic nematodes.According to the cDNA sequences of B1b fragment which was derived from the forward RDA, the gene specific primers were designed. A venom allergen-like protein gene from M. incognita (designated Mi-vap-2) was successfully cloned by RACE and genomic amplification. The gene Mi-vap-2is1917bp long, contains three introns which range in size from39to797bp and four exons range in size from37to361bp. The cDNA of Mi-vap-2contains an open reading frame encoding294amino acids with the first16amino acids being a putative secretion signal. Southern blot analysis suggested that Mi-vp-2is probably the member of a small multigene family. In situ hybridization analyses showed the transcripts of Mi-vap-2accumulated exclusively within the subventral oesophageal gland cells of M. incognita. RT-PCR analyses confirmed Mi-vap-2was transcribed mainly in the pre-parasitic second-stage and early post-inoculation juveniles. All the results indicated that this venom allergen-like protein gene from M. incognita may play an important role in establishment of the parasitic relationship between plants and nematodes.The effectiveness of neurologically compounds of octopamine and resorcinol on stimulating the uptake of infective second-stage juveniles (J2) from M. incognita in vitro was compared. The results showed that octopamine didn’t have effect on stimulating the uptake of J2through stylet and pharynx, whereas resorcinol led to an apparently uptake. Optimization of the conditions for resorcinol to stimulate uptake in vitro by J2was carried out. cDNA of Mi-vap-2from M. incognita was used as template and in vitro transcription was performed to synthesize the VAP dsRNA. RNA interference(RNAi)mediated Mi-vap-2gene silencing was conducted based on the optimized conditions for uptaking. RT-PCR detections confirmed the decrease of Mi-vap-2transcription in nematode samples treated with VAP dsRNA. The reduction in the reproduction ability of nematodes was proved on sensitive tomato plants after dsRNA treatments. The successful RNAi in vitro for Mi-vap-2was demonstrated in this study which validated Mi-vap-2may have an essential function in parasitism of root-knot nematode to plants.The sense and anti-sense cDNA sequences of venom allergen-like protein gene Mi-vap-2from M. incognita were inserted into the vector pHANNIBAL. The transformed clone containing reverse repeat sequences was subcloned into the binary vector pART27and a dsRNA expression construct was produced. Subsequently the construct was introduced into tobacco by leaf-disk method with the help of Agrobacterium tumefaciens. Southern blot analysis showed that the construct was integrated into the genome of tobacco with single copy and obtained stable heredity in T2generation of transgenic lines. Northern blot analysis validated that the transgenic lines produced small interfering RNAs (siRNA) which was homologous to the Mi-vap-2. No obvious reduction in pathogenicity of M. incognita to transgenic tobacco plant was observed, which demonstrated that Mi-vap-2may be not an appropriated target gene for RNAi strategy of controlling root-knot nematodes. The strategy of silencing the target gene in root-knot nematode by expressing specific dsRNA in the host plant was proved to be practical in this study.更多还原