【作者】 蒙姣荣；

【导师】 陈保善； 李华平；

【作者基本信息】 华南农业大学 ， 植物病理学， 2005， 博士

【摘要】 本研究克隆了支持烟草花叶病毒(TMV)和黄瓜花叶病毒(CMV)复制的番茄基因ToTOM3。该基因全长cDNA共有1267个核苷酸,包含一个含888个核苷酸的编码区、72个核苷酸的5′非编码区和307个核苷酸的3′非编码区,编码一个含295个氨基酸、推测是一种七次跨膜蛋白的分子量为34.4 kDa的蛋白质。ToTOM3与拟南芥一个RNA病毒寄主因子基因AtTOM3有76.9%的同源性,与番茄中支持CMV的寄主因子基因ToTOM1有58.6%的同源性。用部分基因组文库结合PCR的方法克隆了ToTOM3基因的全长转录区,共6885bp,包含11个外显子和10个内含子。11个外显子的长度依次为243、91、53、93、72、73、46、64、54、78和400bp。10个内含子的长度依次为2031、155、77、77、654、75、1274、80、333和862 bp。相对定量RT-PCR试验表明ToTOM3基因在番茄子叶期、5叶期、10叶期、结果期等4个生育期的茎、根、叶及结果期的花和果等组织中均有表达,而且表达量基本一致,说明ToTOM3属于组成型表达基因。将ToTOM3基因反义RNA和RNA干扰质粒通过农杆菌介导方法导入番茄,获得26株转基因番茄。Southern杂交表明,这26株转基因番茄的染色体中均携带单拷贝外源基因片段。所有的盆栽转基因番茄均能正常生长、开花和结果。分别用TMV和CMV对5-6叶期转基因番茄和非转基因番茄苗进行接种。用TMV接种10天后,非转基因番茄苗表现出斑驳症状,而转基因苗则无任何症状表现;90天后,接种TMV的非转基因植株仍表现为斑驳症状,而转基因植株无任何症状。苋色藜枯斑定量和DAS-ELISA检测结果显示,所有的转基因植株中TMV均比非转基因对照明显减少,其中,A53中TMV的含量最低,仅相当于对照的7.0%,而A134和R(A)114所含的病毒量最高,相当于对照的52.6%。用CMV接种10天后,非转基因番茄表现出典型的花叶症状,而转基因苗则无任何症状表现;90天后,非转基因番茄苗的叶片严重黄化,形成凹突不平的疱斑,而转基因植株仅表现出轻微的斑驳症状。苋色藜枯斑定量和DAS-ELISA检测结果显示,所有的转基因植株中CMV的含量均比非转基因对照明显减少。不同的转基因株系携带的病毒量有所差异,A25所含的CMV量仅为对照的12.4%,R(A)8为对照的58.1%。高抗TMV的转基因株系同时也高抗CMV。本研究证明,ToTOM3是TMV和CMV共同的寄主因子。同时,本研究也显示,通过抑制寄主因子的表达,可以创造出抗病毒的植物新种质。更多还原

【Abstract】 A homologue of AtTOM3 of Arabdopsis thaliania, designed ToTOM3, was isolated from tomato (Lycopersicum esculentum) and shown to encode a host factor that supports the replication of both Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV).ToTOM3 cDNA was isolated by methods of RT-PCR and 5′- and 3′-rapid amplication of cDNA ends (RACE). The 1267 nt full-length cDNA of ToTOM3 contains an ORF of 888 nt encoding a 295 aa protein with a calculated molecular mass of 34.4 kDa, a 72 nt 5′- and a 307 nt 3′-untranslated regions. The deduced amino acid sequences of the ToTOM3 protein contains several highly hydrophobic regions and is predicted to have seven transmembrane domains. ToTOM3 shares 60.2% identity at nucleotide level and 76.9% identity at amino acid level with that of AtTOM3, which supports the replication of TMV in Arabdopsis. ToTOM3 also shares significant homology (58.6% at amino acid level) with ToTOM1, a gene previously shown to support the replication of CMV in tomato.A genomic fragment of 6885 bp in length that contains the full-length transcribed region of ToTOM3 was isolated by PCR and mini-library screening. As revealed by comparison with full-length cDNA, the ToTOM3 is composed of 11 exons and 10 introns. The size of exons ranges from 46 to 400 bp and the introns from 75 to 2031 bp. All of the exon / intron junction sequences conform to the GT/AG rule. The expression patterns of ToTOM3 were examined by RT-PCR, using tubulin RNA as internal control. It was found that there was not significant difference in mRNA level in different tissues and at different growing stages of tomato.To identify the function of ToTOM3 during the viral infection of the plant host, transgenic tomato plants expressing ToTOM3 antisense RNA and RNAi fragments were generated using Agrobacterium-mediated transformation methods. In the antisense transgenic plants, the full-length transcribed region of the genomic ToTOM3 fragment in reverse orientation was under the control of Cauliflower mosaic virus (CaMV) 35S promoter. And in the RNAi transgenic plants, selected sequences of ToTOM3 cDNA were used to construct a head-to-head inverted repeat that was under the control of CaMV 35S promoter. All of the 26 transgenic tomato plants obtained appeared to grow normally and were able to fruit.The nontransgenic and transgenic tomato plants were inoculated with TMV and CMV. At day 10 post inoculation of TMV, typical mosaic symptoms appeared in the nontransgenic plants. In contrast, no visible symptom was observed in transgenic ones. By day 90 post inoculation, while the nontransgenic plants showing leaf mottling, the transgenic lines remained symptomless. As revealed by both local lesion assay on Chenopodium amaranticolor or DAS-ELISA with TMV-specific antibody, accumulation of TMV in the transgenic plants was significantly lower than that of nontransgenic plants. Similar results were obtained from experiments with CMV infection. The transgenic plants only showed mild symptom 90 days post inoculation, while the control nontransgenic plants showed typical mosaic. The accumulation of CMV in transgenic plants was 12.4 to 58.1% of that of the nontransgenic plants. Transgenic lines that were highly resistant to TMV were also highly resistant to CMV.The above results demonstrate that ToTOM3 is a host factor involved in the replication of TMV and CMV in tomato. This also provided a successful example that resistance to viral infection can be engineered by suppression of a host factor that supports the replication of virus in a major crop.更多还原