【作者】 刘红梅；

【导师】 温孚江；

【作者基本信息】 山东农业大学 ， 植物病理学， 2003， 博士

【摘要】 RNA介导抗病性作为一种获得抗病毒作物的有效策略，因具有抗性表型近乎免疫、抗性持久以及生物安全性高等特点，越来越受到重视，成为植物抗病毒基因工程的研究热点。利用该策略已获得了抗病毒植株，涉及多种病毒和植物。但目前对RNA介导病毒抗性机制还不清楚，RNA介导抗性离实际应用也还有一定距离。本研究的主要内容包括：利用已获得的转非翻译PVY CP(pPVYCPM)不同抗性的烟草,研究外源基因的整合方式与RNA介导病毒抗性的关系;通过不同抗性材料的嫁接试验，对RNA介导抗性的传导规律以及马铃薯Y病毒（PVY）的传播移动规律加以研究。具体结果如下：1．在转pPVYCPM基因烟草Southern blot 分析时发现,同一转基因植株的基因组DNA用不同酶切时所得到的拷贝数不同，如用HindⅢ酶切时M4-1,M74-1, M84-1,M18-1的拷贝数分别为7，6，5，4；而用EcoRI酶切拷贝数分别为2，4，4，5。多数情况下，HindⅢ酶切所得拷贝数多于用EcoRI酶切。特别是当转基因以多拷贝整合时，由于转基因串联重复序列的存在,酶切产生大小相同的带，Southern blot往往不能准确反映转基因的真实拷贝数，因此应选择能产生最多杂交带的酶。重复序列的存在使杂交带间信号强弱不一，为了使结果更精确，确定拷贝数和杂交带的大小时应以放射自显影的X光片为准。2．对转pPVYCPM基因的不同抗性转基因株系T1代的研究表明，转基因的拷贝数与植株的抗病性有一定的联系。高度抗病和抗病植株往往含有多拷贝的转基因，HindⅢ和EcoRI酶切的平均拷贝数为5和3；感病植株拷贝数较少，HindⅢ和EcoRI酶切的平均拷贝数分别为2.3和3.1。这说明拷贝数与抗病性有关，但也有例外如M4-3, M7-1虽然拷贝数较高，但不含串联重复序列，表现为感病。这说明拷贝数并不是决定抗病性的关键因素。3．从已获得的转非翻译PVY CP基因烟草中选取高度抗病和感病株系各4份，对其T1代植株分别进行卡那霉素抗性鉴定、PCR检测和PVYN抗病性鉴定。分别用HindⅢ和EcoRI对不同抗性的T1代植株基因组DNA进行单酶切，以0.8 kb的PVY CP 基因片段作为探针Southern blot分析表明，高度抗病的转基因植株中大多含有转基因的串联重复序列，而感病转基因植株则不含。串联重复分正向重复（direct repeat）和反向重复（inverted repeat）两种类型，抗病转基因植株含有其中之一或二者兼有。说明串联重复序列是决定RNA介导抗性的关键，发生于不同物种上不同诱因的转录后基因沉默（PTGS）最终都以双链RNA（dsRNA）为汇合点,这也是各种形式的PTGS最大的共同点。而多拷贝转基因重复序列能够通过对相邻转基因的通读转录产生这样的dsRNA，而dsRNA被认为是触发PTGS的关键。可见转基因拷贝数对于RNA介导抗性并不是一种剂量效应而是一种质量效应。<WP=10>4．高度抗病株系M13 T0、T1代卡那霉素抗性鉴定、抗病性鉴定和Southen blot分析表明，T1代与T0代相比，卡那霉素抗性和抗病性分离较小，分别为2.1%和 6.2%。T1代杂交带型和拷贝数有较大分离，但多数仍为多拷贝，表现高度抗病。根据高度抗病株系T1代抗病性、卡那霉素抗性分离率较低，遗传背景一致的特点，可以考虑将分子生物学手段与常规育种相结合，对T0代株系进行全面鉴定，筛选多拷贝高度抗病植株进行组培扩繁，将其T1代直接作为品种用于生产。5．单嫁接实验表明，PVY CP介导的抗病性并不能从高度抗病砧木传到上部转基因感病接穗或未转基因的接穗。RNA介导的抗病性是系统性的，一旦产生整株都不发病，这一现象说明植株体内可能存在传导的抗性信号。而病毒在植株体内是通过韧皮部进行长距离传播的，因此抗性信号的被阻断很可能发生于韧皮部。而PVY编码的伴随组分蛋白酶Hc-Pro的主要功能是作用于韧皮部的导管，使其排阻极限增加，协助病毒长距离运输，因此Hc-Pro很可能参与了抗性信号的阻断。6．双嫁接试验证明，PVYN病毒可以跨越一段较长的（可达42cm）高度抗病植株的茎传递到未转基因的接穗中并引起症状，中部高度抗病植株不产生症状，ELISA检测为阴性。病毒能够通过高度抗病的茎段进行长距离运输这一现象说明，韧皮部中的RNA介导抗性可能受到抑制。7．利用农杆菌渗入法对若干转pPVYCPM的感病株系的研究表明，瞬时表达并不能启动RNA介导的抗性机制，对抗性起决定作用的可能还是植物体内的转基因。RNA介导的抗性属于PTGS，但比一般的PTGS更为复杂，因为它涉及病毒进攻和寄主防御的各种机制间的相互作用。更多还原

【Abstract】 RNA-mediated resistance is an effective way to obtain virus resistant plants and has attracted many researchers’ attention because of its advantage of high bio-safety and long duration. RNA-mediated virus resistance is known as pathogen-derived resistance (PDR) and is one kind of posttranscriptional gene silencing (PTGS). PTGS can be induced by transgenes or viral infection and triggers the degradation of RNAs with homology or complemetarity to the foreign genes’ transcript or viral RNA. The signal leading to the degradation of specific RNA can be transmitted. However results from different systems have thrown this simple model into disarray, especially for PTGS signaling. In this research project, tobacco plants transformed with untranslatable PVY CP gene were used to examine the relationship between transgene integration patterns and RNA-mediated resistance more directly . Meanwhile we also investigated the transmission of RNA-mediated resistance and PVY movement on resistant plants and susceptible plants. The main results are as the following: 1. The copy number of foreign genes was always different when then genomic DNA was digested by different restriction endonuclease .The copy number by HindⅢwas always more than that by EcoRI in tobacco. When foreign genes were integrated in multiple copies, Southern blot analysis can’t reflect the real copy number because the fragments generated are of the same size. In order to get accurate results, we digested genomic DNA with the endonuclease that could produce the most copies and copy number should be determined according to X-ray radioautography.2. Southern blot analyses of T1 plants, which acquired different resistance, indicated that the resistance was related to copy number. The resistant plants contained 3-5 copies while the susceptible plants contained 2-3 copies. But M4-3, M7-1 seem to be the only exception, which, though having high copy numbers, were lack of tandem sequences.HindⅢ and EcoRI were used to digest genomic DNAs from the progeny T1 transgenic plants with different response to PVY infection respectively. The results of Southern blot analysis indicated that almost all of the highly resistant plants contained repeat foreign sequences, including direct repeat (DR) and inverted repeat (IR). The highly resistant plants always contained both of or one of these constructs. While susceptible transgenic plants contained neither of them. Research from different systems identified dsRNA as a joint of various forms of PTGS. In cases where dsRNA could form directly, the triggering of the PTGS process may be straightforward. Readthrough transcription of loci of foreign gene repeats could produce a self-complementary RNA<WP=12>3. that formed a duplex consequently. Thus the arrangement of foreign genes in the plant genome plays an important role in the establishment of resistance.4. By single graft experiments, we discovered that RNA-mediated resistance couldn’t be transmitted from highly resistant rootstocks to susceptible scions in spite of the presence of the foreign in the scions. However RNA-mediated resistance is a kind of systemic reaction. Once it occurs, the whole plant becomes immune to virus. This indicated the existence of resistance signals. It is considered that virus move from cell to cell via the plasmodesmata and move over a long distance via phloem. Thus the block of resistance signal might occur in phloem. Consistent with the antiviral nature of RNA-mediated resistance, many viruses use a counter defensive strategy, encoding proteins that block one or more steps in the RNA-silencing pathway. Helper-component-proteinase (Hc-Pro), a viral suppressor of RNA-silencing encoded by plant potyviruses, plays a role in resistant signal blocking by enlarging the size exclusion limit of the phloem. Otherwise the signal from resistant rootstocks may be too little to induce RNA-mediated resistance in susceptible scions. 5. Double graft experiments indicated that PVY could move through a long stem (about 42cm) of high PVY resistance and led t更多还原