【作者】 李建国；

【导师】 郑学勤；

【作者基本信息】 华南热带农业大学 ， 作物遗传育种， 2002， 博士

【摘要】 美洲商陆抗病毒蛋白(Pokeweed Antiviral Protein，PAP)是从美洲商陆(Phytolacca americana)叶片或种子分离出的一类碱性蛋白，分子量29-31KD。PAP是典型的单链核糖体失活蛋白，它可以催化水解原核和真核核糖体的大rRNA3’-端茎环结构(stem-loop)中的腺嘌呤残基，导致核糖体失活。PAP可以有效抑制若干种非相关病毒，包括植物病毒和动物病毒，利用PAP基因可以通过转单基因获得对多种病毒具有广谱抗性的转基因植株。成熟的PAP有262个氨基酸，其中靠C端的25个氨基酸为毒性区，PAP的C端缺失突变体PAP-c没有毒性区域，它的细胞毒性很低，但仍具有较强的抗病毒活性。 本研究采用PCR技术获得PAP和PAP-c的cDNA，同时利用番茄叶片特异表达启动子rbcS-3A，构建不同的植物表达载体，通过农杆菌介导的遗传转化体系将PAP和PAP-c基因转入番茄自交系TP002，获得转基因植株，是植物抗病毒基因工程研究领域一次新的探索；获得的转基因番茄植株对TMV和CMV病毒具有抗性，可以作为番茄育种的一个新的抗源材料。 主要研究结果如下： 1．采用PCR技术，分离并克隆到美洲商陆抗病毒蛋白cDNA(PAP)和C-端缺失突变体cDNA(PAP-c)。克隆到的PAP编码区为940bp，与Genebank中的序列同源率为99.7％；克隆到的PAP-c编码区为775bp，与相应的序列同源率为99.6％。 2．采用PCR技术，从番茄基因组中分离并克隆到rbcS-3A启动子。测序结果表明，克隆到的基因为1150bp(包括酶切位点)，与Genebank中的核苷酸序列同源率为99％，具有完整的TATA box和CAAT box，在增强子、沉默子和启动子功能区域与Genebank中的相应序列同源率为100％。 3．利用克隆到的基因和植物表达载体pBI121，成功构建了3个美洲商陆抗病毒蛋白的植物表达载体：pBPAP，pBPAP-c，pB-rbc-PAP-c。其中，pB-rbc-PAP-c为植物特异表达载体，启动子为番茄rbcS-3A启动子，在叶片中受光诱导表达，具有组织特异性。 4．通过农杆菌介导的遗传转化体系分别将2个植物表达载体pBPAP和pB-rbc-PAP-c导入番茄，经PCR检测和Southern检测，得到3株转基因番茄植株。RT-PCR检测结果表明，在CaMV 35S启动子的作用下，PAP基因在转基因番茄(9#植株)叶片和果实中均可以表达；在rbcS-3A启动子的作用下，PAP-c基因只在转基因番茄(6#植株)叶片中表达而不在果实中表达。人工接种病毒结果显示，与对照植株相比，5#和9#转基因植株对TMV具有抗性，6#转基因植株对TMV和CMV具有抗性。更多还原

【Abstract】 Pokeweed antiviral protein(PAP), isolated from leaves and seeds of Phytolacca americana, are single-chain ribosome-inactive proteins(RIPs) with molecular mass of 29 to 31KD, which can inhibit translation by catalytically removing a specific adenine residue from a highly conserved, surface-exposed, stem-loop structure in the large rRNA of eukaryotic and prokaryotic ribosomes. PAP displays broad-spectrum antiviral activity against a number of different viruses, including plant and animal viruses. The property of PAP provides a way to produce multiple-virus-resistant transgenic plants by introducing and expressing a single gene. The C-terminal deletion mutant, PAP-c, which loses 25 amino acids near C-terminus comparing to the mature PAP of 262 amino acids, has lower cytotoxicity and shows resistance to viruses. These 25 amino acids near C-terminus is necessary for toxicity but not for antiviral activity, suggesting that antiviral activity of PAP can be dissociated from its toxicity. Expression of PAP-c in transgenic plants will be safer than that of wild-type of PAP.According to the published sequence of PAP, the cDNA encoding PAP and PAP-c were obtained by using PCR, respectively. The leaf specific expression promoter rbcS-3A, which confers light-inducible and organ-specific expression, was also amplified from tomato DMA by PCR. In order to transfer PAP or PAP-c gene into tomato, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 358 promoter and rbcS-3A promoter respectively. Agrobacterium tumefaciens containing plant transformation vectors was used to transform tomato self-line TP002 by the leaf disc method.The results of this study are as follows:1 The cDNA encoding PAP and C-terminal deletion mutant PAP-c were obtained by using PCR. The PAP cDNA has an open reading frame of 940bp, which is 99.7% homologous to the published sequence. The PAP-c cDNA has an open reading frame of 775bp, which is 99.6% homologous to the corresponding sequence of the published PAP cDNA.2 The rbcS-3A promoter sequence was amplified from tomato genome DNA by using PCR. Sequencing results showed, the 1150bp fragment obtained in this study has 99% homology to the rbcS-3A promoter sequence published in GeneBank and contains intact TATA box andCAAT box. Its enhancer region, silencer region and promoter function region are 100% homologous to those of published rbcS-3A.3 Based on pBI121, three plant-expression-vectors containing PAP or PAP-c were constructed with CaMV 35S promoter and rbcS-3A promoter respectively. The pBPAP has PAP gene expressed from CaMV 35S promoter and pBPAP-c has PAP-c gene with the same promoter. The pB-rbc-PAP-c has PAP-c gene modulated by rbcS-3A promoter from tomato.4 By Agrobacterium tumefaciens mediated transformation, PAP and PAP-c gene were induced into tomato self-line(TP002) through two different plant expression vectors, pBPAP and pB-rbc-PAP-c, respectively. Three regenerated tomato plants were proved to be transgenic plants by PCR and Southern blotting. The results of RT-PCR for transgenic plants showed, PAP gene was expressed in both leaves and fruits of No.9 transgenic plant under CaMV 35S promoter, PAP-c gene was only expressed in the leaves not in the fruits of No.6 transgenic plant under rbcS-3A promoter. To test the resistance to virus of transgenic tomato plants, they were mechanically inoculated with TMV and CMV respectively. The results indicated , comparing to the non-transformed control plants, No.5 and No.9 plants displayed resistance to TMV, No.6 plant displayed resistance to TMV and CMV.更多还原