【作者】 吴廷全；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 Harpin蛋白是由植物病原细菌产生的一类多功能激发子,能够促进植物生长和增强植物抗病、抗虫和抗逆能力。本论文的研究内容之一是对水稻细条斑病菌(Xanthomonas oryzae pv. oryzicola)产生的蛋白质激发子HpaGXooc及其三个功能片段HpaG1-94、HpaG10-42、HpaG62-138(下标数字表示在HpaGXooc中的氨基酸序列起始和终止位置)对绿茶产量和品质所产生的效应展开研究。田间试验结果表明HpaG1-94、HpaGXooc和HpaG10-42,尤其是HpaG1-94,能够显著提高绿茶的产量和品质。对其内在分子与生化机制的研究显示HpaG1-94和HpaGXooc不仅调控绿茶中生长相关基因与品质相关基因的转录表达,而且促进儿茶酚的大量积累。核黄素(VB2)也是一种多功能的激发子,它对人类、动物、植物及微生物的生长和抗病具有重要的作用。植物和多种微生物能够在体内合成核黄素,而人类和动物只能从食物中获得。外源施用核黄素能够诱导乙烯信号、茉莉酸信号和防卫反应信号,从而提高植物生长与抗病能力。对于增加内源核黄素的含量会对植物产生哪些生物效应并不清楚。而2,4-二氧四氢喋啶合酶(lumazine synthase, LS)是催化核黄素生物合成途径中倒数第二步的酶,是核黄素合成的关键酶之一。本论文的研究内容之二是将从水稻中克隆的2,4-二氧四氢喋啶合酶基因(oryza sativa Lumazine Synthase, OsLS)分别转入烟草和水稻,获得了异源表达的转基因烟草和同源过表达的转基因水稻。对转基因植物进行研究的结果显示这两种转基因植物在增加内源核黄素含量和增强抗病性方面是一致,但生长存在差异；同时,我们对转基因植物生长和抗病的分子机制进行了初步探讨。突变体是研究基因功能最有效的材料之一。随着生命科学和生物技术的迅猛发展,获得突变体的方法和手段也多种多样。而转移DNA (transferred DNA, T-DNA)插入突变以其技术成熟、操作简单和便于分析而在植物科学研究中获得广泛应用。本论文研究的第三个内容是通过T-DNA插入的方法得到了对烟草花叶病毒(tobacco mosaic virus, TMV)和大白菜软腐病菌(Erwinia carotovora pv. carotovora, ECC)具有抗病性的121-3突变体,并通过一系列分子生物学方法对其进行了分析,最终,得到了一段与植物抗病性有关的植物与T-DNA边界相融合的序列。HpaGXooc及其三个功能片段对绿茶的生物学效应HpaGXooc是由水稻条斑病菌(Xanthomonas oryzae pv.oryzicola)产生的一类harpin蛋白质激发子。为了优化其功能,本实验室通过基因改造和蛋白原核表达的方法获得了HpaGXooc的九种不同大小的功能蛋白片段。实验室前期的研究表明HpaG1-94、HpaG10-42以及HpaG62-138,均比HpaGXooc能够更好地促进番茄和烟草的生长、抗病和诱导过敏性细胞死亡(hypersensitive cell death, HCD)。本研究是将HpaGXooc及其三种功能片段HpaGXooc和HpaG62-138)在一种重要的饮品植物-绿茶(Camellia sinensis)上施用,试验研究结果表明分别施用HpaGXooc、HpaG10-42和HpaG62-138的茶树与施用空载体对照(empty vector protein, EVP)(不含HpaGXooc及其功能片段)的茶树相比,能够提高茶叶产量和品质,改良茶树生化特性。特别是施用HpaG1-94, HpaGXooc的茶树与对照相比,它们的采茶时间提前5-16天,采茶次数均多出一次；茶头数分别比对照增加38.3%和36.1%；茶叶鲜重分别比对照增重50.5%和37.3%；茶叶干重分别比对照增重55.8%和42.3%。此外,施用HpaG1-94的茶树与对照相比,上等茶的比例提高30.8%,茶叶的悬浮时间也较长。儿茶酚是一种有利于人体健康的抗氧化剂,它是决定茶叶色、香、味的重要成分,其含量是衡量茶叶品质的最重要的生化指标。我们的研究结果表明,这四种蛋白均可以大幅度提高茶叶中儿茶酚的含量(64.5%-118.8%)。为了更进一步阐述HpaGXooc及其功能片对茶树影响的分子机制,我们选取了综合表现较好的HpaG1-94和HpaGXooc进行了基因水平的研究。在分别施用了HpaG1-94、HpaGXooc和EVP的茶树芽头中,我们检测了植物生长相关的expansins基因(CsEXPL1、CsEXPL10和CsEXPL18)和茶叶品质相关基因(CsCHS、CsDFR、TCS1和CsSAMS)的转录表达情况。CsCHS、CsDFR、TCS1和CsSAMS分别是儿茶酚、花色素苷、咖啡碱生物合成中的关键酶。我们的实验结果表明,HpaGXooc和HpaG1-94均可以增强expansins基因和CsCHS, CsDFR基因在绿茶芽头中的表达,且HpaG1-94促进这些基因表达水平的能力明显强于HpaGxooc. expansins基因的增强表达与提高绿茶芽头的生物量有一定的关系,而CsCHS, CsDFR基因表达量的增加对茶叶的色、香、味等品质的提高起一定的作用。而TCS1和CsSAMS在HpaGXooc和HpaG1-94处理过的茶树芽头中的表达比对照有所减弱,这样可以使茶叶保持适度的咖啡碱含量,有利于茶叶品质的提高。综上所述,我们认为HpaG1-94能够更有效地提高绿茶的产量和品质。2、水稻2,4-二氧四氢喋啶合酶对植物的生物学效应核黄素(VB2)也是一种多功能的激发子,它对人类、动物、植物及微生物的生长和抗病具有重要的作用。植物和多种微生物能够在体内合成核黄素,而人类和动物只能从食物中获得。外源施用核黄素能够诱导乙烯信号、茉莉酸信号和防卫反应信号,从而提高植物生长与抗病能力。对于增加内源核黄素的含量会对植物产生哪些生物效应并不清楚。2,4-二氧四氢喋啶合酶(LS)是催化核黄素生物合成途径中倒数第二步的酶,是核黄素合成的关键酶之一。本研究从水稻(oryza sativa)品种中花11中克隆到2,4-二氧四氢喋啶合酶基因,定名为OsLS (oryza sativa Lumazine Synthase). OsLS基因mRNA全长为666bp,编码一个含有221个氨基酸的蛋白质,其氨基酸序列与高等植物具有较高的同源性(68%～75%),而与微生物的同源性较低,仅有32～49%。对LS的氨基酸进化分析显示水稻与其它高等植物具有更近的亲缘关系,而与微生物的亲缘关系较远。在大肠杆菌内表达OsLS和His-tag的融合蛋白,大小约为30kDa,其中OsLS,约23 kDa; His-tag,约7kDa. OsLS蛋白不能引起烟草叶片的过敏性反应(Hypersensitive Response, HR)。同时,我们利用蛋白质亚细胞定位预测软件targetp v1.1对水稻OsLS进行预测,预测结果显示OsLS定位于叶绿体的可靠性高达0.879。随后,我们用胶体免疫金(ImmunoGold)与Western blot两种方法对转基因烟草检测和用ImmunoGold对野生型和转基因水稻检测的结果均证实了这一预测。为了深入解析OsLS对植物的生物学效应,我们将OsLS基因转入烟草(三生烟)和水稻(中花11),获得了异源表达OsLS的转基因烟草(LSETT)和同源过表达的转基因水稻(LSOETR),并对它们进行了分子验证(Southern blot, RT-PCR, Western blot).同时,我们还设计了转基因沉默载体,但转化水稻后未能获得转OsLS基因沉默的水稻苗,推测出现这一现象的原因是OsLS基因的沉默对植物具有致死作用,这与先前报道的细菌和真菌无法产生LS突变体的原因是LS突变体对微生物具有致死作用的结论是一致的。表达OsLS的转基因烟草(LSETT)与野生型(WT)相比,其营养生长加快,对烟草花叶病毒(tobacco mosaic virus, TMV)的抗性增强。对LSETT的生长相关基因NtEXP1、NtEXP2、NtEXP6和抗病防卫相关基因NPR1、PR-1a、PR-1b、GST1进行检测的结果显示NtEXP1、NtEXP2、NPR1、PR-1a、PR-1b. GST1的表达与WT相比显著增强,与其生长和抗病表型相一致。WT和LSETT分别同时接种TMV后于不同时间点取烟草的系统叶片并用3,3-二氨基联苯胺(diaminobenzidine, DAB)检测叶片中的活性氧,试验结果表明OsLS能够消弱烟草由于受到胁迫而积累的活性氧(ReactiveOxygen Species, ROS),这有利于使植物细胞免于受过度氧化的伤害。近年来的研究认为,合适的活性氧浓度对诱导植物抗病性是有利的,而过高浓度的活性氧对植物具有相当大的伤害作用。hinl和hsr203是HCD标志性基因,我们对其检测的结果表明它们在LSETT中的表达水平比WT的要弱,这可能与植物体内活性氧的积累量有关。另外,我们测定了LSETT和WT中的黄素(Flavins)、乙烯(ethylene)、茉莉酸类(JAs)的含量,测定的结果是这三者在LSETT中的含量均高于WT中的。其中,黄素(Flavins)包括游离态核黄素(riboflavin, VB2)、黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)和黄素腺嘌呤单核苷酸(flavin mononucleotide, FMN),茉莉酸类包括茉莉酸(jasmonic acid, JA)和茉莉酸甲酯(methyl jasmonate, Me-JA).同时,在接种TMV后不同时间点取样,我们检测了乙烯通路和茉莉酸通路中的一些基因如ETR1, CTR1, ERF1, EREBP1,COI1,结果表明,LSETT与WT相比,其乙烯信号途径和茉莉酸信号途径均有所增强。对过量表达OsLS的转基因水稻(LSOETR)的研究与转基因烟草的研究方法和思路类似。在水稻苗龄达120天时,检测LSOETR叶片中riboflavin、FAD、FMN的含量均比野生型的高。LSOETR与对照相比,其分蘖较少,长势较差,控制分蘖的基因OsMOC1与生长旺盛标志性基因OsGRF1的表达低于野生型水稻,且其抽穗滞后15天左右。然而,当水稻苗龄达157天时,LSOETR的株高却远远超过野生型,其穗也较长,但其分蘖数依然很少。当接种水稻白叶枯病菌Xanthomonas oryzae pv.oryzae)PXO99, LSOETR较WT表现出较好的抗病性,其病程相关蛋白基因OsPR1b与OsPR10受到强烈诱导表达,而对照的表达相对较弱。与野生型水稻相比,LSOETR与LSETT一样能够消除活性氧的积累。综上所述,水稻OsLS对植物具有多种重要的生物效应,特别是在抗病和生长方面。3、烟草抗病突变体(121-3)的研究突变体是研究基因功能最有效的材料之一。随着生命科学和生物技术的迅猛发展,获得突变体的方法和手段也多种多样。近年来,由于农杆菌转化方法的不断完善,T-DNA插入突变以其技术成熟、操作简单和便于分析而在植物科学研究中获得广泛应用,产生了大量的植物突变体,并建立起许多T-DNA插入突变体库,其中研究较多的植物是拟南芥、烟草、水稻和大麦.本研究用农杆菌介导的T-DNA插入的方法对三生烟草进行转化,获得了120个转化成功的品系,并用烟草花叶病毒(TMV)和大白菜软腐病菌(ECC)分别接种,幸运地筛选到抗TMV、ECC的烟草突变体121-3品系。并通过热不对称交错聚合酶链式反应(thermal asymmetric interlaced polymeri polymerase chain reaction, TAIL-PCR)得到T-DNA右边界侧翼植物序列(281bp)。此段植物序列在WT中并不转录表达,而在121-3突变体中被激活表达,且此段植物序列能被TMV和ECC诱导增强表达。因此：.我们推测,此段植物序列的转录可能与植物的抗病性有关。此外,我们通过3’末端cDNA快速扩增PCR (3’rapid amplification of cDNA ends PCR,3’-race PCR)和5’末端cDNA快速扩增(5’rapid amplification of cDNA ends PCR,5’-race PCR)的方法在121-3突变体中获得了植物序列两端的序列。我们所获得的植物序列位于T-DNA负链右边界的侧翼。T-DNA负链的部分序列能够与其相连的侧翼植物序列融合转录表达。此外,与野生型相比,121-3突变体中的JAs含量显著提高,ERF1基因表达明显增强,这暗示了121-3突变体可能与乙烯信号和茉莉酸信号有关系。至于,侧翼植物序列与这些信号通路和抗病性的关系还有待于进一步研究。4.创新点1)筛选出能够大幅度提高绿茶儿茶酚含量和产量的HpaG1-94功能蛋白片段；2)从单子叶植物中克隆到OsLS基因,并深入研究其生物学功能,得到了具有抗病性的转基因水稻和烟草,这是增加植物内源核黄素来提高植物防卫反应的一个很好的实例；3)用T-DNA插入方法产生了具有广谱抗病性的烟草突变体,对其研究表明,且T-DNA负链右边界与侧翼植物序列融合转录表达,并可能与植物抗病性有关。更多还原

【Abstract】 Harpin proteins produced in plant pathogenic bacteria can enhance plant growth and increase plant resistance to diseases, insect pests, and envirometal stresses. HpaGxooc, a harpin protein from Xanthomonas oryzae pv. oryzicola, the pathogen that causes bacterial leaf streak in rice. Fragments HpaG1-94, HpaG10-42, and HpaG62-138, contain HpaGXooc regions of amino acid sequence as indicated by the number spans. Here, we reported productivity and biochemical properties of green tea (Camellia sinensis) in response to the fragments and HpaGxooc tested in comparison with an inactive protein control and HpaG1-94. HpaGxooc and HpaG10-42, especially HpaG1-94, have better effects on increasing quantity and improving quality of green tea leaves. In addition, HpaG1-94 and HpaGxooc not only regulate transcriptional expression of genes of growth-related and quality-related, but increase accumulation of catechol in green tea leveas.Riboflavin (VB2) is also a multifunctional elicitor and it plays a key role on growth and disease resistance of man, animals, plants, and microorganisms. Despite essentiality of vitamin B2, however, only plants and certain microorganisms can synthesize it themselves, whereas higher animals including man must obtain it through their diets. Exogenous application of riboflavin can induce ethylene signaling, J A signaling, and defense reaction, which promotes plant growth and increases resistance to disease. But what effects does accumulation of endogenous riboflavin in plant has on plants? It is unknown. And Lumazine synthase which catalyzes the penultimate step of riboflavin biosynthesis is one of key enzymes of riboflavin biosynthesis. Here, we cloned lumzaine synthase gene (OsLS) from rice (oryza sativa) strain Zhonghua11 and transformed it into plant callus cells and transgenic tobacco plants of heterologous expression of OsLS and transgenic rice plants of homologous overexpression of OsLS were gained. Studies on transgenic plants with OsLS gene indicated that the two transgenic plants are similar in accumulation of endogenous riboflavin and disease resistance, but they are different in growth. Moreover, we explored molecular mechanism of growth and disease resistance of the two kind of transgenic plants initially.Mutant is one of most appropriate materials studying Gene functions. And with the rapid development of biological science and technology, the methods of producing mutants are more and more. But, the technique of T-DNA insertion is used widely in plant science because of mature technique, simple operation and convenient analysis. In this study, we gained 121-3 mutant resistant to TMV and ECC by T-DNA insertation and investigated the mutant by molecular biological methods, and eventually, fused sequence of flank plant sequence and border sequence of T-DNA was gained and it was related with plant disease resistance.1. Biological effects of green tea in response to full-length and three functional fragments of HpaGxoocHpaGxooc, a harpin protein elicitor, was produced by a pathogenic bacterium (Xanthomonas oryzae pv. oryzicola) that can cause bacterial leaf streak in rice. To optimize the biofunctions of HpaGXooc, nine functional fragments containing different numbers of amino acid were produced by gene reconstruction and prokaryotic expression in our laboratory. Our previous studies showed that three functional fragments HpaG1-94, HpaG10-42, and HpaG62-138, which contain HpaGXooc regions of amino acid sequence as indicated by the number spans, can enhance growth, increase disease resistance, and induce Hypersensitive cell death (HCD) of tomato and tobacco plants more strongly than HpaGXooc. Here, we report that HpaGxooc and its functional fragments (HpaG1-94, HpaG10-42, HpaG62-138) are applied to green tea(Camellia sinensis), a famous drink plant. And our research results indicate that productivity and biochemical properties of green tea (Camellia sinensis) in response to the three fragments and HpaGxooc are tested in comparison with an inactive protein control and the results show HpaG1-94, HpaGXooc, and HpaG10-42 have better effects on increasing quantity and improving quality of green tea leaves. Especially, compared with the control, the period of picking tea of green tea plants treated with HpaG1-94 and HpaGXooc respectively is 5-16 days earlier, one time more,38.3%and 36.1%increased more in the number of tea buds,50.5%and 37.3%increased more in fresh weight of tea,55.8%and 42.3%increased more in dry weight of tea. In addition, HpaG1-94 can elevate the proportion of tea of high quality to reach 30.8%and prolong tea leaf floating time. Catechol, an antioxidant, which is good to human health, is an important compound which affects tea quality such as colour, aroma, savor, and so on. The content of catechol in green tea is a critical biochemical criterion of the quality of green tea. Our study results indicated HpaGxooc and its three functional fragments all elevated the contents of catechol in green tea, and in contrast to the control, the contents of catechol was added to 64.5%-118.8%. To expatiate on the molecular mechanism of HpaGxooc and its functional fragments affecting green tea, HpaG1-94 and HpaGxooc have many good exhibitions in above research results and therefore the two proteins are selected to carry out the study of gene levels. To reveal the molecular basis for plant growth enhancement, we detected transcriptional expression of tea (Camellia sinensis) expansion-like genes (CsEXPLs) including CsEXPL1, CsEXPL10 and CsEXPL18, which regulate growth of the cell and the plant, and tea quality-related genes including CsCHS, CsDFR, TCS1 and CsSAMS, which were key enzymes of biological synthesis of catethol, anthocyanin, caffeine and theine respectively. The results of our studies indicate that HpaG1-94 and HpaGxooc can elevate transcriptional expression of CsCHS, CsDFR and CsEXPLs in green tea buds, and effect ofHpaG1-94 is much stronger than that of HpaGxooc.Enhancement of expression of CsEXPLs favors increase of biomass of green tea buds, and the induced expression of CsCHS and CsDFR is in favor of the formations of quality of colour, aroma and savor in green tea. However, decrease of expression of TCS1 and CsSAMS in tea buds treated with HpaG1-94 and HpaGxooc is beneficial to control caffeine and theine at moderate and correct levels, which can increase tea quality. Overall, HpaG1-94 much exceeds the full length HapGxooc protein to increase yields and improve the quality of green tea.2. Biological effects of lumazine synthase from rice on plantsRiboflavin (VB2) is also a multifunctional elicitor and it plays a key role on growth and resistant diseases of man, animals, plants, and microorganisms. Despite essentiality of vitamin B2, however, only plants and certain microorganisms can synthesize it, whereas higher animals, including man must obtain it through their diet. Exogenous application of riboflavin can induce ethylene signaling, JA signaling and defense reaction, and enhances plant growth and increase resistance to disease. But what effects does accumulation of endogenous riboflavin in plant has on plant? It is unknown. And Lumazine synthase which catalyzes the penultimate step of riboflavin biosynthesis is one of key enzymes of riboflavin biosynthesis. Here, we cloned lumzaine synthase gene from rice (oryza sativa) strain Zhonghua11,and it was named OsLS. Complete sequence of mRNA of OsLS gene contains 666 base pairs (bp) which encodes a protein containing 221 amino acids, and its amino acid sequence is 68-75% identical with the amino acid sequences of lumazine synthases from higher plants however, OsLS is only 32-49%identical at the amino acid sequence level to its counterparts in microorganisms. A phylogenetic tree is built from the alignment of OsLS and its putative orthologues in several microorganisms and plant species and the result shows OsLS has a much closer relative to those in other higher plants than LSs in microorganisms. OsLS protein with His-tag was expressed in E.coli and Molecular mass of the fusion Protein was predicted to be about 30kD (OsLS, about 23 kD; His-tag, about 7 kD). OsLS protein (100mg/ml) can not elicit hypersensitive response (HR). At the same time, we used localization prediction software, targetp v1.1, to gain the predicted result of OsLS localization, which the reliability of this protein localized in chloroplast is up to 0.879. To test the hypothesis that OsLS is a chloroplast protein, we demonstrated it by determining transgenic tobacco using Western blot and ImmunoGold and determing transgenic rice using ImmunoGold.To understand biological effects of OsLS protein on plants further, OsLS gene was transformated into tobacco (xanthi) and rice (zhonghuall) respectively, and transgenic tobacco plants (LSETT) of heterologous expression of OsLS gene and transgenic rice plants (LSOETR) of homologous overexpression of OsLS gene were gained and these transgenic plants were identified by Southern blot, RT-PCR and Western blot. In addition, transgenic silencing vector of OsLS gene was constructed to transform rice cell, but no transgenic rice plants were produced and this might be as a result that OsLS gene silencing was lethal to plants, which was consistent with previous publications that OsLS gene silencing was lethal to bacteria and fungi and therefore no mutant of OsLS gene silencing was produced. In comparison to wild type tobacco plants, transgenic tobacco plants with OsLS gene have better growth and much stronger disease resistance to tobacco mosaic virus (TMV). Plant growth-related genes such as NtEXPl, NtEXP2 and NtEXP6, and defence response genes such as NPR1, PR-1a, PR-1b and GST1 were determined in LSETT and WT and the results indicated that these genes except NtEXP6, are all induced to express stronger in LSETT, which was consistent with growth and disease resistance of LSETT. Systemic leaves of LSETT and WT were stained with diaminobenzidine (DAB) to determine reactive oxygen species (ROS) in different time points after inoculation with TMV and the experimental result indicated that OsLS had the ability to eliminate ROS produced in plants under the condition of oxygen stress, which could make plant cell to keep away from damage from overoxidation. In recent years, the researchers have a general view that proper concentration of ROS is benefical to induce plant disease resistance and too high concentration of ROS is very harmful to plant. hin1 and hsr203, which are the maker genes of hypersensitive cell death (HCD), were investigated to indicate that their expression levels were also lower in LSETT than in WT, which might have relative to accumulation of ROS in plants. And that we determined the contents of flavins, ethylene (ET) and jasmonates (JAs) in leaves from LSETT and WT respectively, and the results indicated that compared to WT, the levels of the tested substances were all higher in LSETT leaves. Thereinto, Flavins include free riboflavin (vitainm B2, VB2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and JAs include jasmonic acid (JA) and methyl jasmonate (MeJA). At the same time, these genes, ETR1, CTR1, ERF1, EREBP1 and COI1 in ethylene signaling pathway and jasmonic acid signaling pathway were determined in different tome points postinoculation with TMV and their expression levels were changed, which showed ET and JA signalings were activated much strongly respectively, in LSETT.Research way and method on transgenic rice overexpressing OsLS (LSOETR) were similar with those on transgenic tobacco. Riboflavin, FAD, and FMN were determined in leaves when rice growed to 120d, and in comparison to wild type, the levels of riboflavin, FAD, and FMN in LSOETR were much higher. But LSETR grows much worse and has fewer tillers than the control, and the levels of gene expression of OsMOC1, which controls the number of tiller, and OsGRF1, related to growth, were both weaker than the control, and the heading date of rice of LSOETR was postponed 15d. However, LSOETR growed much higher and its spikes were much longer but its tillers were till fewer than the control after transplanted 157d. Compared to wild type rice, LSOETR behaved much stronger disease resistance after inoculating PXO99 (Xanthomonas oryzae pv.oryzae) and pathogenisis-related genes, OsPR1b and OsPR10 were activated to express much stronger. In comparison to wild type rice, LSOETR can delete accumulation of ROS, the same as LSETT. Clearly, OsLS from rice has multiple biological effects on plant, especially in plant growth and disease resistance.3. Studies on tobacco resistant disease mutantMutant is one of most appropriate materials studying Gene functions. And with the rapid development of biological science and technology, the methods of producing mutants are more and more. In recent years, because method of agrobacterium tumefaciens-mediated transformation is perfect continually, the technique of T-DNA insertion is used widely in plant science in virtue of mature technique, simple operation and convenient analysis. Large numbers of plant mutants were produced by T-DNA insertion and many T-DNA insertion mutant banks have been constructed, and mutants were from arabidopsis, tobacco, rice, and barley. In this study, tobacco (xanthi) cells were transformed by T-DNA insertion of agrobacterium tumefaciens-mediated and 120 transgenic tobacco strains were produced, and these transgenes were inoculated with tobacco mosaic virus (TMV) and Erwinia carotovora pv.carotovora (ECC) respectively, fortunately, we screened a tobacco mutant strain named 121-3 resistant to TMV and ECC. Subsequently, we gained flank plant sequence (281bp) of T-DNA right border by thermal asymmetric interlaced PCR(TAIL-PCR). In wild type tobacco (WT), the flank plant sequence was not transcripted, but it was activated to transcript in 121-3 mutants. And expression of the plant sequence was induced in 121-3 mutants by TMV and ECC, but not in WT. Therefore, we thought that the flank plant sequence was related with disease resistance. Furthermore, both-side sequences of the plant sequence were gained by 3’-race and 5’-race. We found that the flank plant sequence was localizaed at negative strand of T-DNA and partial sequence of negative strand of T-DNA and the flank plant sequence were fused to transcript. In addition, our results indicated that compared to WT, in 121-3 mutant, the levels of JAs and transcription accumulation of ERF1 gene both were increased remarkably, which suggested 121-3 mutant may relate to ethylene signaling and JA pathways. The relations of the flank plant sequence and these signaling pathways and disease resistance are indispensable to study further.4. Conclusive remarks1) HpaG1-94 protein fragment was selected because it not only increased plant yields, but also, most of all, elevated catechol content in green tea.2) OsLS gene was cloned from monocotyledon rice and was transformed into rice and tobacco repectively. Transgenic rice and transgenic tobacco exhibited better resistant to disease, which was a good example in increasing endogenous riboflavin to induce plant defense reaction.3) Tobacco broad-spectrum disease resistant mutant was generated by T-DNA insertion and our studies showed partial sequence of negative strand of T-DNA and the flank plant sequence were fused to transcribe and it may be related with plant disease resistance.更多还原