生物激发子与氧化还原相关信号对植物生长和抗病性的调控作用
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摘要
随着人们对农作物上使用农药的关心，寻找一种安的保护农作物的方法是全世界努力的方向。生防细菌和其他一些天然来源的材料在病害防治和提高产量上有着巨大的应用潜力。Pseudomonas和Bacillus species是一类非常重要的生防细菌，以多种机理保护植物，也能促进种子发芽和植物生长。Harpin是多种植物病原细菌产生的Ⅲ型效应因子的蛋白质，在病原菌侵染的时候泌出激发植物的多种反应，外源施用到植物上能引起多种有利效应。HpaG_(xoo)，Xanthomonas oryzae pv．oryzae产生的一种harpin蛋白质，能激发植物对病原菌和昆虫的防卫反应，促进植物生长，hpaG_(xoo)的转基因烟草系统获得抗性(systemic acquired resistance，SAR)受到诱导。生防细菌在根部的定殖能诱导植物的ISR(induced systemic resistance，诱导系统抗性)，ISR与SAR拮抗，赋予植物截然不同的抗病机理。因此，Ⅲ型效应因子和生防细菌一起使用很可能比其中一个单独使用对植物具有更好的诱导作用。本研究的目的是探清生防细菌和Ⅲ型效应因子是如何互作的，如何影响植物的生长和抗病性。P．cepacia P6854和B．subtilisB-916对水稻纹枯病有比较好的防效，我们产生了hpaG_(xoo)的转基因水稻(品种R109)的一些株系，研究发现HpaG_(xoo)-expressing rice line 1 (HER1)生长较快，对盐胁迫和病原菌的防卫反应较强。
Elicitin是疫霉菌产生的一类寄主特异性蛋白质激发子，能诱导多种烟属植物、萝卜和芸苔产生过敏反应(hypersensitive response，HR)，但对番茄、马铃薯等茄科植物，多数十字花科植物等无效。Elicitin不仅能诱导植物的过敏反应，也能诱导多种植物的多种防卫反应，例如：可以诱导烟草抗黑胫病(P．parasitica)和由Xanthomonascampestris pv armoraciae引起的病害，也能使烟草抗TMV的侵染。Harpins和elicitins这两类重要的激发子，诱导植物抗病、抗逆的机理既有相似之处，又有不同，二者共同作用于植物时，是否能协同作用，需要进一步研究。在本研究中构建了能同时表达两种激发子的工程菌，研究了该双元激发子的工程菌的蛋白粗提液与单元激发子蛋白粗提液引起烟草的微敏反应和系统抗病性的差异。
核黄素(VB_2)在细菌和高等生物内是核黄素结合辅酶FMN和FAD的重要成分，FMN和FAD是主要的酶促反应如氢化物、氧和电子传递反应的酶的辅酶因子，是一种多功能性的维生素，对动植物、微生物的生长、抗病都有很重要的作用。核黄素在植物和微生物中都能自我合成，合成途径的倒数第二步是由2，4-二氢喋啶合酶(lumazine synthase，LS)催化的，倒数第一步是由核黄素合酶(riboflavin synthase，RS)催化的。动物和人类都缺少这两种酶，但是很多细菌和酵母却依赖这两种酶形成内源核黄素，很久以来LS和RS蛋白质高级结构得到很好的研究，用来设计疫苗，治疗人类和动物的疾病，很多细菌和酵母编码LS和RS蛋白质的基因已经得到克隆。在拟南芥中发现LS与茉莉酸途径相交叉，与根对茉莉酸的敏感性、植物的抗病性密切相关。由此可推断水稻上的OsLS和OsRS很可能存在着目前不为所知的功能，在植物上编码这两个酶的基因的报道不多，尤其是水稻的这两个基因根本还没得到克隆。
硫氧还蛋白(thioredoxin，TRX)是一种小分子量蛋白质，催化巯基-二硫键的交换反应，参与细胞还原环境的调节，在不同的组织和器官内有不同的构象，具有多功能性。目前已经在多种微生物、植物和动物内克隆到该基因，细胞质、叶绿体和线粒体内都有多种构象的硫氧还蛋白，除了能消除氧胁迫，这种蛋白质越来越多的功能被发现。烟草上该基因(NtTRX)的功能研究甚少，在生长和抗病性上的功能尚未见报道。
本博士论文的研究目的就是初步研究清楚激发子HpaG_(xoo)与生防细菌P．cepacia和B．subtilis或者激发子ParAl一起使用时对植物的生长、抗病性和过敏反应的影响是否存在互作；水稻的核黄素合成过程的酶促步骤的OsLS和OsRS基因和烟草的其中一个氧化还原作用的NtTRX基因(NtTRXh)在植物的生长、抗病性、细胞死亡和活性氧胁迫上的作用。研究结果如下：
1生物激发子与生防细菌协同作用影响檀物的生长、抗病和过敏反应
为了搞清生防细菌和HpaG_(Xoo)在作物上一起存在时是否能够对植物的生长和抗病有更好的作用，研究了P．cepacia和B．subtilis在野生型水稻R109和表达hpaG_(xoo)的水稻株系HER1上的作用。与野生型R109相比，HER1的长势较好、产量高、对病害和盐胁迫的抗性强。与水处理的对照相比，P．cepacia和B．subtilis的定殖促进R109和HER1根的生长，也能促进R109茎叶的生长，但是HER1茎叶的生长受到抑制。接种纹枯病菌(Rhizoctonia solani)后用P．cepacia和B．subtilis处理，R109和HER1的发病程度都比对照轻，而且HER1有更好的抗性，这说明P．cepacia、B．subtilis和HpaG_(xoo)在诱导植物的抗病性上相互协作。P．cepacia和B．subtilis在根部定殖后，HER1和R109上一些与生长和防卫密切相关的基因具有不同的表达谱。在R109的根内，调控植物生长的OsARF1表达量与P．cepacia和B．subtilis对生长的促进作用是一致的，相反在HER1的茎叶内，OsARF1表达量与P．cepacia和B．subtilis对水稻的抑制作用相一致。在R109和HER1内参与植物生长的编码延展蛋白的OsEXP1对P．cepacia的反应在茎叶内与生长一致，但是在根内却不一致。OsMAPK编码细胞分裂素激活的蛋白质激酶，调控水稻内对盐和病菌侵染的防卫反应，P．cepacia和B．subtilis处理后，R109内OsMAPK的早期表达与抗病性是一致的，但是HER1受P．cepacia处理还是不处理，OsMAPK的表达量都是相似的。显然P．cepacia、B．subtilis和HpaG_(xoo)在水稻的生长和抗病性上的互作是不同的，然而P．cepacia、B．subtilis和HpaG_(xoo)在抗病性方面的协作在农业生产上具有巨大的应用潜力，生防细菌、Ⅲ型效应因子和病原菌的互作原理值得深入研究。
ParAl和hpaG_(xoo)基因同时连接到表达载体pET30a(+)上，构建二元重组质粒pET30a(+)：：parAl：：hpaG_(xoo)，转化BL21(DE3)，生成工程菌株BL21：：parAl：：ApaG_(xoo)。Tris-Tricine缓沖系统电泳发现，有两条15 kDa和10 kDa的目的蛋白。不煮或煮沸半小时处理蛋白质溶液然后注射枯斑型三生烟叶片观察是否引起过敏反应，得到能同时表达寄生疫霉激发子ParAl和白叶枯病菌激发子HpaG_(xoo)的工程菌。同时表达二者的BL21：：pET30a(+)：：parAl：：hpaG_(xoo)的蛋白粗提液(ParAl：：HpaG_(Xoo)与HpaG_(xoo)和ParAl相比，引起较强烈的烟草微敏反应，效应基因hin1和hsr203表达强烈，诱导烟草强烈的对花叶病毒的系统抗性，病程相关基因(PR1a和PR1b)表达较强烈。由此可见，能同时表达HpaG_(xoo)和ParA1两类激发子的工程菌在农业生产上可能具有广阔的应用前景。
总之，激发子HpaG_(Xoo)能与生防细菌P．cepacia、B．subtilis和激发子ParA1互作，影响植物的生长、抗病性和过敏反应。
2水稻的两个基因OsLS和osRS的功能
本研究首次克隆到分别催化水稻核黄素合成过程的倒数第二步和倒数第一步的关键酶的编码基因OsLS和OsRS。OsLS基因全长666 bp，在氨基酸序列上与拟南芥(Arabidopsis thaliana)、菠菜(Spinacia oleracea)和烟草(Nicotiana tabacum)的LS基因的同源性非常高，在68-76％之间，根据氨基酸序列分析绘制系统进化树，在同一个进化分枝内。与稻瘟病菌(Magnaporthe grisea)和发光细菌(Photobacteriumphosphoreum)的核苷酸序列完全没有同源性，氨基酸序列有一定的同源性，分别是33％和48％，根据氨基酸序列分析绘制系统进化树，在不同的进化分枝内。在大肠杆菌内能表达出包括His-tag在内的约29．85 kDa大小的OsLS蛋白质。该蛋白质有221个aa，分子量是22，471．44 Da，蛋白质等电点是10．01，是一种碱性蛋白质。远远高于激发子HpaG_(xoo)蛋白质溶液的浓度(100μg／ml)的OsLS蛋白质水溶液注射烟草叶片不能引起过敏反应，这说明该蛋白质本身对植物的细胞没有损害。软件分析该蛋白质定位在叶绿体内，与拟南芥、菠菜和烟草的LS蛋白质的定位相同，OsLS蛋白质单体有5个α螺旋，4个β折叠，5个α螺旋把4个β折叠包围在中间。OsRS序列全长1662 bp，与Gene ID OJ1111H02．Predgene04的不含内含子和非翻译区的序列(no intronsequence and no other untranslated regions)完全一致。NCBI Blast分析，OsRS基因与A．thaliana(拟南芥基因库内的推测序列)、Candida albicans、C．famata、Filobasidiellaneoformans、Sinorhizobium meliloti、Bartonella elizabethae、Saccharomyces cerevisiae、Pichia guilliermondii、P．Phosphoreum、Schizosaccharomyces pombe、E．coli等的RS基因在核苷酸序列和氨基酸序列上都没有同源性，在系统进化树的不同进化分枝内。在大肠杆菌内表达该蛋白质，能表达包括His-tag在内的约66．37 kDa大小的蛋白质。100μg／ml的OsRS蛋白质水溶液注射烟草叶片不能引起过敏反应，这说明OsRS蛋白质本身对植物的细胞没有损害。软件分析该蛋白质有553个氨基酸，分子量是59，589．80 Da，等电点是6．00，酸性蛋白质，定位在叶绿体内，该蛋白质的折叠结构有6个α螺旋，5个β折叠，6个α螺旋把5个β折叠作为核心包围在中间。
OsLS和OsRS转基因烟草的游离态的核黄素、FAD和FMN含量都有所升高，而且OsLS转基因的烟草(LST)核黄素含量略高于OsRS转基因烟草(RST)的核黄素含量。LST和RST比转空载体的烟草对照植株(VECT，CK)营养生长旺盛，幼苗期表现更强烈，能使细胞壁疏松、促进植物生长的expansin genes在LST和RST内表达水平比CK表达水平高。LST和RST与对照相比细胞编程死亡和细胞死亡的标志基因hinl和hsr203的表达没有显著差异。与对照相比LST和RST抗病性增强，而且LST的抗病性强于RST。在接种TMV之前，LST和RST的抗病相关基因PR1a、PR1b的表达量都远远高于对照烟草内的表达量，接种TMV之后的同样时间，LST和RST内的PR1a、PR1b都受到诱导表达，受诱导的量远远大于对照烟草内受诱导的量，而且LST内PR1a和PR1b的整体表达水平高于RST内的表达水平。乙烯是重要的介导生长和防卫反应的信号分子，释放量增多能引起植物的生长加快和防卫反应增强。LST和RST比对照烟草乙烯的产生速度快、产生量大，RST在开始的乙烯产生量高于LST，但是LST随着时间变化增长幅度比RST大，LST和RST的生长和抗病的不同表现可能与这种乙烯的释放量有关。活性氧是一种与植物的编程性细胞死亡和抗病性密切相关的信号因子，低浓度的活性氧诱导植物的细胞死亡和抗病性，但是高浓度的活性氧会破坏植物的细胞膜，使植物丧失对病害的抗性。转基因的烟草在不接种病毒的时候，活性氧的产生量与对照烟草活性氧的产生量没有差异，接种病毒后48小时差异增大，对照烟草受到的活性氧的胁迫大于LST和RSt，而且LST活性氧产生量略小于RST，这种消除活性氧的胁迫的差异应该与抗病性的差异有关。
构建了OsLS和OsRS的过表达载体和沉默载体，转化水稻幼胚愈伤组织，成功得到OsLS和OsRS过表达的水稻转基因株系，OsgS部分沉默的水稻株系。OsLS发生过表达、完全或部分沉默很可能都对水稻的分化和生长产生严重的影响，转基因过程中发现，OsLS和OsLS片段的发夹结构转化水稻，愈伤组织开始分化比空载体转化的愈伤组织分化迟，但是OsLS片段的发夹结构转化的愈伤组织开始出现绿色后不能继续分化，逐渐褐化死亡。OsLS发生沉默可能是一个致死的过程，这与前人的研究细菌和真菌无法产生LS的突变体在一定程度上是一致的。OsRS转化的愈伤组织分化早，成苗快，OsRS片段的发夹结构转化的愈伤组织分化比空载体转化的愈伤组织分化迟，比OsLS转化的愈伤组织分化早。4个月苗龄的OsRS和OsLS过表达的水稻的游离态核黄素、FAD、FMN都比对照植株转化空载体的水稻(VECR)高；OsRS发生部分沉默的植株(SiRS)与VECR相比核黄素含量降低。OsRS转基因水稻(RSR)与VECR相比，分蘖多、长势好，控制分蘗的基因OsMOC1和生长活跃的标志基因OsGRF1表达量大大高于对照水稻内的表达量，抽穗时间大大提前。SiRS与VECR相比，分蘗较少、长势稍差，OsMOC1和OsGRF1表达量略低于对照水稻内的表达量，抽穗时间稍推迟。OsLS过表达株系(LSR)与对照相比，其分蘗能力和生长势大大下降，OsMOC1和OsGRF1表达量少于对照水稻内的表达量，抽穗时间大大延迟。三种株系的细胞死亡没有明显差异，也即OsLS和OsRS不影响植株的衰老。接种白叶枯病菌PX099，LSR对白叶枯病的抗性最好，病程相关基因OsPR1b和OsPR10受到强烈的诱导，RSR的抗性有小幅提高，OsPR1b和OsPR10受到较强烈的诱导，弱于LSR；SiRS的抗性明显降低，抗病性几乎完全丧失，OsPR1b和OsPR10几乎不受到诱导表达。与对照水稻相比，LSR和RSR受到的活性氧的胁迫减小，而且OsLS的抑制作用强于OsRS，但是SiRS受到的氧胁迫大于对照植株，较低的合适浓度的活性氧对植物的抗病性是有利的，但是过高浓度的活性氧会大大损害植物细胞。
总之，水稻的OsLS和OsRS具有非常重要的作用，分别对抗病和生长有很强的促进作用，都能消除活性氧的胁迫，而且OsLS的消除作用强于OsRS。这两个基因对植物的编程性细胞死亡都没有影响。OsLS在烟草和水稻上对生长的作用不同的原因值得深入研究。这两个基因在水稻的育种中有很重要的应用前景。
3烟草NtTRXh基因负调控营养生长，正调控抗病性，与细胞死亡无关
克隆到烟草(Nicotiana tabacum)的thioredoxin h-like protein complete CDS序列，该蛋白具有完整的TRX蛋白质的活性位点WCGPC。序列的同源性和系统进化树分析可见，NtTRX-h1(AF435818)与小麦的Tal(AF438359)、大麦的Hvh1(AF435815)和拟南芥的Ath-t1(AAG51342)中的氨基酸序列同源性非常高，分别达到71％、72％和78％，在同一个进化簇内；但是与番茄(Lycopersicon esculentum)的LeCITRX-pt(AF261142)和Solanum tuberosum的StCDSP32(Y09987)的氨基酸序列同源性很低，分别为30％和24％，在不同的进化簇内：与烟草(N．alata)的Nah(DQ021448)的氨基酸序列同源性也不高，为37％，在不同的进化簇内。在大肠杆菌内表达该蛋白质，能表达包括His-tag(大约5，426 Da)在内的约22，260 Da大小的蛋白质。150μg／ml的TRX蛋白水溶液不能引起烟草叶片的过敏反应，这说明该蛋白质本身对植物的细胞没有损害。软件分析该蛋白质有152个aa，分子量是17，022．20(17．02 kDa)，蛋白质等电点(pI)是4．53：很可能是一种线粒体基质空间内和细胞质内的蛋白质，微粒体和线粒体内膜上可能少量存在(或不存在)；TRX蛋白的高级构象有2个α螺旋，5个β折叠。
成功构建该基因到病毒沉默植物载体pBinPlus2β：：1．7A上，得到pBinPlus2β：：1．7A：：trx，转化农杆菌EHA105，注射烟草，部分沉默ZRX基因的转录表达。成功构建该基因到含有激发子诱导性启动子的载体pBI121：：PPP1上，得到pBI121：：PPP1：：TRX，转化农杆菌EHA105，叶盘法转化烟草，产生转基因的烟草PPP1：：TRX，以空载体pBI121：：PPP1转化烟草做对照。使用激发子harpin_(Ea)诱导转基因烟草发现，PPP1在受到harpin_(Ea)诱导后，能成功引起烟草内源基因TRX的过表达。TRX基因发生部分沉默的植株(siTRX)营养生长速度都比对照植株快，达到显著性差异，NtEXP1、NtEXP2和NtEXP6基因表达量增长；相反TRX基因发生过表达的植株(OvXRX)营养生长速度都比对照植株(CK)慢，达到显著性差异，NtEXP1、NtEXP2和NtEXP6基因表达量略有下降。SiTRX、OvTRX分别和对照植株相比细胞死亡没有明显差异，细胞死亡标志基因hin1和hsr203的表达水平也没有明显差异。植株的TRX基因发生部分沉默对FMV的抗性明显降低，病斑数大大增多，感病程度升高，达到显著性差异，抗病防卫反应受到严重损害，抗病相关基因PR1a和PR1b在TRX发生沉默但是没有病毒侵染的情况下，表达量高于非沉默植株，这对植物本身不利，造成内源物质的浪费，但是一旦发生TMV病毒侵染，非沉默植株的基因表达量在24小时后迅速高于沉默植株，也就是TRX基因可能干扰了PR基因的诱导表达，相反植株TRX基因发生过表达对TMV的抗性明显升高，病斑数大大减少，抗病性增强，达到显著性差异，抗病性增强，抗病相关基因PR1a和PR1b在TRX发生过表达时一旦发生TMV病毒侵染，OvTRX的基因表达量在24小时后迅速高于对照植株，也就是TRX基因可能增强了PR基因的表达。活性氧与植物的抗病性密切相关，高水平的活性氧产生会使植物迅速丧失抗病能力。接种TMV后，SiTRX和OvTRX活性氧的产生在前24小时与对照植株都没有差异，但是从第48小时，SiTRX的活性氧产生量大大多于CK，大量的活性氧损害了PR基因的诱导表达，破坏了烟草的抗病机制，相反OvTRX的活性氧产生量大大低于CK的产生量，植株受到的氧胁迫降低，抗病性增强。总之，TRX基因抑制烟草的营养生长，诱导抗病性，不影响编程性细胞死亡。全文总结
通过上述研究，我们对激发子HpaG_(xoo)与生防细菌和ParA1的互作、水稻核黄素合成过程的酶2，4-二氢喋啶合酶和核黄素合酶、烟草的硫氧还蛋白对植物的生长发育和抗病性的作用有了初步的认识。首先，激发子HpaG_(xoo)能与生防细菌P．cepacia、B．subtilis和激发子ParA1互作，影响植物的生长、抗病性和过敏反应。其次，水稻的OsLS和OsRS具有非常重要的作用，OsLS对抗病性有很强的诱导作用，OsRS对生长具有很强的促进作用，二者都能消除活性氧的胁迫，而且OsLS的消除作用更强烈，这两个基因对植物的编程性细胞死亡都没有影响。这两个基因在水稻的育种中有很重要的应用前景，有利于培育生长周期短、产量高、抗病性好的品种。最后，TRX基因抑制烟草生长，诱导抗病性，不影响编程性细胞死亡。
本研究为开发更优化的病害防治策略，培育具有优良生长和抗病性状的品种，研究核黄素信号途径与氧化还原信号在植物的生长和抗病性上的信号网络和目标蛋白的研究准备了坚实的理论基础和转基因材料的物质基础，下一个要解决的问题是寻找这些基因在植物内的目标蛋白和相交叉的信号通路，探索这些通路之间的互作网络。
Due to public concerns on pesticide use in crops, exploration on alternative methods has been a global effort to secure crop. Biocontrol bacteria and natural products from various sources show a great potential of agricultural use in disease control and crop product improvement. Pseudomonas and Bacillus species is an important class of bioeontrol bacteria; and they protect plants with many mechanisms. Moreover, biocontrol bacteria can promote seed germination and plant growth with indirectly increasing disease resistance. Plant responses are induced by the biocontrol bacteria colonization on plant roots and play a role in disease control depending on the response speed and magnitude in contrast to the infection to plants by pathogens. Natural products which have a potential in crop improvement are various in nature and sources. HpaG_(Xoo), a harpin produced by Xanthomonas oryzae pv. oryzae, stimulates plant growth and resistance against pathogens and insects. Moreover, the biocontrol bacteria colonization on plant roots activates induced systemic resistance(ISR), which antagonizes SAR and provides plants with a distinct battery of defense arsenal. Therefore, a combinative use of biocontrol bacteria and the type-Ⅲeffectors could be more effective than use of either of them in crop improvement. Previously, we used P. cepacia P6854 and B. subtilis B-916 to control rice sheath blight with desired results. We have introduced HpaG_(Xoo) into rice(variety R109), generating several transgenic lines. They were improved in disease resistance. Here we show that the HpaG_(Xoo)-expressing rice line 1(HER1) increases growth and activates defenses toward salinity and pathogens. We present evidence that P. cepacia P6854 and B. subtilis differentially affects growth and disease resistance in R109 and HER1.
Elicitins are a peculiar of proteins produced by oomycetes in the genera Phytophthora and Pythium, and ParA1 is 10 kDa elicitor produced by P. parasitica var. nicotinanae, host special elicitor, sensitive to heat and protease K in vitro. Elicitins induce not only plant hypersensitive response(HR) but also defense reponses. In order to get more effective and more economical recombinant bacteria than the recombinant E. coli BL21(DE3) that expressing ParA1 and HpaG_(Xoo) singly, we constructed a bacterium expressing HpaG_(Xoo) and ParA1 simultaneously. The protein of the bielicitor bacterium has been tested for its effect on tobacco micro-HR and the resistance against tobacco mosaic virus.
Riboflavin is an important ingredient of flavin mononucleotide(FMN) and flavin adenine dinucleotide(FAD) in bacteria and eukaryotic organisms. FMN and FAD participate in many enzyme catalizing reactions, so riboflavin is a multifunctional vitamin which is essential for the maintenance of life. The two enzymes, lumazine synthase and riboflavin synthase, catalyze the last two steps in the biosynthesis of riboflavin. The two enzymes represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with the enzymes of the mammalian metabolism. The genes of many bacteria and yeasts have been cloned, but there are only little reports on plants and no research on rice. It has nearly been reported that the cos1 mutation(LS mutation) restores the coil-related phenotypes, including defects in JA sensitivity, senescence and plant defense responses in Arabidopsis, but there have no research about the functions on growth, disease resistance, active oxygen and hypersensitive cell death of the two genes of rice by so far. It is worth to research.
Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cells. The plant thioredoxin system is particularly complex since many thioredoxin isoforms are found in plants and they are multifunctional proteins. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. The functions of tobacco thioredoxin h-like protein have not been reported, we try to research this gene actions on plant growth and disease resistance.
This study is focused on crosstalk between representative and distinct agencies that induce plant defense and growth, and functions of pivotal regulators in redox-based signal transduction that regulates both effects in plants. The interactions of HpaG_(Xoo) with P. cepacia or ParA1, and functions of the genes encoding lumazine synthase and riboflavin synthase from rice, and a gene encoding h-like thioredoxin from tobacco, as well, have been studied by multiple methods.
1 Bioelicitor and PGPR interact to affect plant growth and disease resistance Expression of HpaG_(Xoo), a bacterial type-Ⅲeffector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG_(Xoo) can act together to provide better results in crop improvement. We studied effects of P. cepacia and B. subtilis on the rice variety R109 and the hpaG_(Xoo)-expressing rice line HER1. Compared to R109, HER1 increased growth, grain yield and defense responses toward diseases and salinity stress. Colonization of roots by P. cepacia or B. subtilis caused some increase, in contrast to controls, in root growth of R109. Growth of R109 leaves and stems and HER1 roots were effected a little but leaves and stems of HER1 was inhibited. When P. cepacia and B. subtilis colonization was subsequent to plant inoculation with Rhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting that P. cepacia or B. subtilis and HpaG_(Xoo) cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding to P. cepacia or B. subtilis. In R109 roots, the OsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion by P. cepacia or B. subtilis. Inversely, OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression of OsEXP1, which encodes an expansin protein involved in plant growth, was concomitant with growth in leaves and roots, in response to P. cepacia or B. subtilis. We also studied OsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response to P. cepacia or B. subtilis., an early expression of OsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whether P. cepacia was present or not. Evidently, P. cepacia or B. subtilis and HER1 interact differently in rice growth and resistance. Whereas combinative effects of P. cepacia or B. subtilis and HpaG_(Xoo) in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-Ⅲeffectors and pathogens.
HpaG_(Xoo) and ParA1 are two important elicitors produced by plant pathogens, the genes parA1 and hpaG_(Xoo) were cloned by polymerase chain reaction and ligased into the expressing vector pET30a(+), transformed into E. coli BL21(DE3). The protein of BL21:: parA1:: hpaG_(Xoo) (ParA1:: HpaG_(Xoo)) was extracted, and electrophoresed by Tris-Tricine SDS-PAGE, 15 kDa and 10 kDa two proteins were got. The ParA1 and HpaG_(Xoo) were identified expressing synchronously because of different macroscopic hypersensitive response(HR) of tobacco leaves injected with the proteins boiled 30 min or not. ParA1:: HpaG_(Xoo) induced stronger microscopic hypersensitive response(micro-HR), systemic acquired resistance(SAR) to tobacco mosaic virus and expression of pathogen related genes, PR1a and PR1b than ParA1 or HpaG_(Xoo) only did.
2 The roles of lumazine synthase and riboflavin synthase in plant growth and defense
Based on transgenic and molecular studies, the functions of both enzymes from rice were researched in this study.
OsLS and OsRS are first cloned. OsLS have 666 nucleotides. The nucleotide sequence identities of LS genes of Oryza sativa, Arabidopsis thaliana, Spinacia oleracea and Nicotiana tabacum are between 68-76％, and the genes are in the same branch of phylogenetic tree. The nucleotide sequences of LS genes of Magnaporthe grisea and Photobacterium phosphoreum have no identity, but the amid acid sequences of them have 33％and 48％identities and the genes are in different branches of phylogenetic tree. OsLS protein with His-tag(29.85 kDa) was expressed in E. coli. OsLS protein has 221 aa, its molecular weight is 22, 471.44 Da, and pI is 10.01.100μg/ml OsLS protein aqueous solution cann't induce tobacco hypersensitive response, meaning this protein is not bad for plants. The protein, analyzed online, is localized in chloroplast, as the same as the location of the LS proteins of A. thaliana, spinach and tobacco. Its tertiary structure has 4μfolds, surrounded by 5αhelixes. OsRS has 1662 nucleotides, having 100％identity with Gene ID OJ1111H02. Predgene04. NCBI Blast analyed that OsRS has no identity with the RS sequences of A. thaliana, Candida albicans, C. famata, Filobasidiella neoformans, Sinorhizobium meliloti, Bartonella elizabethae, Saccharomyces cerevisiae, Pichia guilliermondii, P. phosphoreum, Schizosaccharomyces pombe and E. coli, and the genes are in different branches of phylogenetic tree. OsRS protein with His-tag was expressed in E. coli, whose molecular weight is 66.37 kDa. 100μg/ml OsRS protein aqueous solution can't induce tobacco hypersensitive response, meaning this protein is not bad for plant. It has 553 aa, the molecular weight is 59, 589.80 Da and pI is 6.00. The protein analyzed online is localized in chloroplast, and its tertiary structure has 5βfolds, surrounded by 6αhelixes.
OsLS and OsRS trangenic tobacco have higher levels of free riboflavin, FMN and FAD than that of the blank vector transgenic tobacco(VECT), and the OsLS transgenic tobacco (LST) have higher level than OsRS transgenic ones(RST). LST and RST have better growth in infancy and youthful times than the wild type(WT) and VECT. Expansin genes, which enloose cell wall and enhance plant growth, of LST and RST have higher expression levels than VECT and WT. RST growed better than LST. OsLS and OsRS have no effect on programmed cell death in transgenic tobacco lines compared with WT and VECT, the marker genes hin1 and hsr203 have similar expression levels in them. The disease resistances of LST and RST were enhanced, the pathogenesis-related genes(PR) were induced stronger than control(CK), and LST have better resistance than RST. Ethylene is an important signal molecular related to development and disease resistance. LST and RST can produce more ethylene than CK, this may resulted into fast growth and strong disease resistance. Active oxygen is important for plant disease resistance, and the active oxygen of high level destroy plant disease resistance. Without TMV inoculation, LST and RST have no difference with CK, but LST and RST were haunted by less active oxygen stress, and especially LST has the least one, this means OsLS has sronger energy to eliminate active oxygen stress than OsRS.
Rice immature embryo calli were transformed with the overexpressing vector and hairpin unit of OsLS and OsRS by soaking the plant material with an appropriate Agrobacterria tumefaciens EHA105 suspension. OsLS and OsRS overexpressing rice(LSR and RSR) and OaRS partly silencing lines(SiRS) have been got, but OsLS haipin transformed rice(SiLS) was not got. The rice calli transformed by OsLS hairpin rice couldn't differentiate. The calli transformed by OsLS overexpressing vector rice grew and differentiated slower than those transformed by the blank vector, but the rice calli transformed by OsRS overexpressing vector grew and differentiated fast. The rice calli transformed by OsRS hairpin vector grew and differentiated slower than CK. Four-month old LSR and RSR have higher levels of free riboflavin, FAD and FMN than CK. SiRS have lower levels of riboflavin, FAD and FMN than CK. RSR have more tillers than CK, but LSR and SiRS have fewer tillers than CK. OsMOC1, which controls tiller formation and OsGRF1, which expresses in actively growing parts, have higher expression levels in RSR than CK, but have lower expression in LSR and SiRS than CK. All lines have no marked difference in programmed cell death. The disease resistance of LSR was enhanced the most among RSR, VECR, SiRS and LSR. RSR were enhanced a little disease resistance than VECR. The defenee response of SiRS were damaged absolutely. The expression levels of pathogenesis-related genes, OsPR1b and OsPR10, were in the same trend with rice blight resistance. LSR and RSR were effected by less active oxygen stress than VECR, but SiRS haunted by more active oxygen stress than CK. So OsLS and OsRS are very important, OsLS induces disease resistance and inhibits growth, but OsRS induces growth and disease resistance, they all eliminate active oxygen stress.
3 Tobacco thioredoxin signaling inhibits plant growth but induces disease resistance and does not affect programmed cell death
Nicotiana tabacum thioredoxin h-like protein complete CDS sequence was cloned, with the conserved active domain WCGPC. The amino acid sequence identities of TRX with those of N. tabacum(NtTRX-hl, AF435818), Triticum aestivum(Tal, AF438359), Hordeum vulgare(Hvhl, AF435815) and Arabidopsis thaliana(Ath-tl, AAG51342) are 71％, 72％and 78％respectively, and the genes are in the same branch of phylogenetic tree. The identities with those of Lycopersicon esculentum(LeCITRX-pt, AF261142), Solanum tuberosum(StCDSP32, Y09987) and N. alata(Nah, DQ021448) are only 30％, 24％and 37％respectively, and the genes are localized in the different branches of phylogenetic tree. TRX protein was expressed with His-tag(5, 426 Da) in vitro, the molecular weight is 22, 260 Da. 150μg/ml TRX protein couldn't induce tobacco hypersensitive response, meaning the protein is not bad to plant. TRX protein has 152 aa, its molecular weight is 17, 022.20 Da, and its pI is 4.53. This protein is most possible in mitochondrial matrix space, and may be in cytoplasm, microbody(peroxisome) or mitochondrial inner membrane. TRX tertiary structure has 2α-helixes and 5β-folds.
TRX partially silenced tobacco plants(SiTRX) grew faster than the control(CK), the difference is marked, and the expansin genes, which enloose cell wall and enhance plant growth, NtEXP1, NtEXP2 and NtEXP6 have higher expression levels than CK. TRX overexpressing plants(OvTRX) have the reverse results compared with SiTRX. TRX silence and overexpression have no effect on hypersensitive cell death compared with CK, the marker genes hin1 and hsr203 had similar expression levels in LST, RST and CK. The disease resistance of SiTRX was damaged, the pathogenesis-related genes PR1a and PR1b were induced less than CK, and this means TRX disturbs these genes expression, in the other side OvTRX have better TMV resistance and higher pathogenesis-related gene expression levels than CK. Without TMV inoculation and within 48 inoculation, active oxygen levels were similar in SiTRX, OvTRX and CK, but 48 hours postinoculation SiTRX produced more active oxygen than CK, but OvTRX produce less than CK. Clearly, the TRX gene inhibits plant growth but induces disease resistance and doesn't affect hypersensitive cell death.
Conclusion
In a conclusion, we have some results about the interaction of HpaG_(Xoo) with P. cepacia, B. subtilis or ParA1, the functions of lumazine synthase and riboflavin synthase from rice and thioredoxin from tobacco. First, there are some interactions between HpaG_(Xoo) and P. cepacia, B. subtilis. or ParA1, which effected on plant disease resistance and hypersensitive response. Second, OsLS and OsRS have important roles on plant growth and disease resistance. OsRS enhances growth, OsLS strongly induces disease resistance, and the two genes doesn't affect programmed cell death. The two genes have important potentials on the culture of the varieties of short-time cycle, high yield and strong disease resistance. Finally, TRX inhibits tobacco growth, induces disease resistance and doesn't affect hypersensitive cell death.
The theory bases and transgenic plants are prepared to explore better plant protection strategy, and to culture the varities with better growth and disease resistance than CK, to study the roles of riboflavin pathway and redox signals in plant disease resistance and growth, and to look for their target proteins.
Elicitin是疫霉菌产生的一类寄主特异性蛋白质激发子，能诱导多种烟属植物、萝卜和芸苔产生过敏反应(hypersensitive response，HR)，但对番茄、马铃薯等茄科植物，多数十字花科植物等无效。Elicitin不仅能诱导植物的过敏反应，也能诱导多种植物的多种防卫反应，例如：可以诱导烟草抗黑胫病(P．parasitica)和由Xanthomonascampestris pv armoraciae引起的病害，也能使烟草抗TMV的侵染。Harpins和elicitins这两类重要的激发子，诱导植物抗病、抗逆的机理既有相似之处，又有不同，二者共同作用于植物时，是否能协同作用，需要进一步研究。在本研究中构建了能同时表达两种激发子的工程菌，研究了该双元激发子的工程菌的蛋白粗提液与单元激发子蛋白粗提液引起烟草的微敏反应和系统抗病性的差异。
核黄素(VB_2)在细菌和高等生物内是核黄素结合辅酶FMN和FAD的重要成分，FMN和FAD是主要的酶促反应如氢化物、氧和电子传递反应的酶的辅酶因子，是一种多功能性的维生素，对动植物、微生物的生长、抗病都有很重要的作用。核黄素在植物和微生物中都能自我合成，合成途径的倒数第二步是由2，4-二氢喋啶合酶(lumazine synthase，LS)催化的，倒数第一步是由核黄素合酶(riboflavin synthase，RS)催化的。动物和人类都缺少这两种酶，但是很多细菌和酵母却依赖这两种酶形成内源核黄素，很久以来LS和RS蛋白质高级结构得到很好的研究，用来设计疫苗，治疗人类和动物的疾病，很多细菌和酵母编码LS和RS蛋白质的基因已经得到克隆。在拟南芥中发现LS与茉莉酸途径相交叉，与根对茉莉酸的敏感性、植物的抗病性密切相关。由此可推断水稻上的OsLS和OsRS很可能存在着目前不为所知的功能，在植物上编码这两个酶的基因的报道不多，尤其是水稻的这两个基因根本还没得到克隆。
硫氧还蛋白(thioredoxin，TRX)是一种小分子量蛋白质，催化巯基-二硫键的交换反应，参与细胞还原环境的调节，在不同的组织和器官内有不同的构象，具有多功能性。目前已经在多种微生物、植物和动物内克隆到该基因，细胞质、叶绿体和线粒体内都有多种构象的硫氧还蛋白，除了能消除氧胁迫，这种蛋白质越来越多的功能被发现。烟草上该基因(NtTRX)的功能研究甚少，在生长和抗病性上的功能尚未见报道。
本博士论文的研究目的就是初步研究清楚激发子HpaG_(xoo)与生防细菌P．cepacia和B．subtilis或者激发子ParAl一起使用时对植物的生长、抗病性和过敏反应的影响是否存在互作；水稻的核黄素合成过程的酶促步骤的OsLS和OsRS基因和烟草的其中一个氧化还原作用的NtTRX基因(NtTRXh)在植物的生长、抗病性、细胞死亡和活性氧胁迫上的作用。研究结果如下：
1生物激发子与生防细菌协同作用影响檀物的生长、抗病和过敏反应
为了搞清生防细菌和HpaG_(Xoo)在作物上一起存在时是否能够对植物的生长和抗病有更好的作用，研究了P．cepacia和B．subtilis在野生型水稻R109和表达hpaG_(xoo)的水稻株系HER1上的作用。与野生型R109相比，HER1的长势较好、产量高、对病害和盐胁迫的抗性强。与水处理的对照相比，P．cepacia和B．subtilis的定殖促进R109和HER1根的生长，也能促进R109茎叶的生长，但是HER1茎叶的生长受到抑制。接种纹枯病菌(Rhizoctonia solani)后用P．cepacia和B．subtilis处理，R109和HER1的发病程度都比对照轻，而且HER1有更好的抗性，这说明P．cepacia、B．subtilis和HpaG_(xoo)在诱导植物的抗病性上相互协作。P．cepacia和B．subtilis在根部定殖后，HER1和R109上一些与生长和防卫密切相关的基因具有不同的表达谱。在R109的根内，调控植物生长的OsARF1表达量与P．cepacia和B．subtilis对生长的促进作用是一致的，相反在HER1的茎叶内，OsARF1表达量与P．cepacia和B．subtilis对水稻的抑制作用相一致。在R109和HER1内参与植物生长的编码延展蛋白的OsEXP1对P．cepacia的反应在茎叶内与生长一致，但是在根内却不一致。OsMAPK编码细胞分裂素激活的蛋白质激酶，调控水稻内对盐和病菌侵染的防卫反应，P．cepacia和B．subtilis处理后，R109内OsMAPK的早期表达与抗病性是一致的，但是HER1受P．cepacia处理还是不处理，OsMAPK的表达量都是相似的。显然P．cepacia、B．subtilis和HpaG_(xoo)在水稻的生长和抗病性上的互作是不同的，然而P．cepacia、B．subtilis和HpaG_(xoo)在抗病性方面的协作在农业生产上具有巨大的应用潜力，生防细菌、Ⅲ型效应因子和病原菌的互作原理值得深入研究。
ParAl和hpaG_(xoo)基因同时连接到表达载体pET30a(+)上，构建二元重组质粒pET30a(+)：：parAl：：hpaG_(xoo)，转化BL21(DE3)，生成工程菌株BL21：：parAl：：ApaG_(xoo)。Tris-Tricine缓沖系统电泳发现，有两条15 kDa和10 kDa的目的蛋白。不煮或煮沸半小时处理蛋白质溶液然后注射枯斑型三生烟叶片观察是否引起过敏反应，得到能同时表达寄生疫霉激发子ParAl和白叶枯病菌激发子HpaG_(xoo)的工程菌。同时表达二者的BL21：：pET30a(+)：：parAl：：hpaG_(xoo)的蛋白粗提液(ParAl：：HpaG_(Xoo)与HpaG_(xoo)和ParAl相比，引起较强烈的烟草微敏反应，效应基因hin1和hsr203表达强烈，诱导烟草强烈的对花叶病毒的系统抗性，病程相关基因(PR1a和PR1b)表达较强烈。由此可见，能同时表达HpaG_(xoo)和ParA1两类激发子的工程菌在农业生产上可能具有广阔的应用前景。
总之，激发子HpaG_(Xoo)能与生防细菌P．cepacia、B．subtilis和激发子ParA1互作，影响植物的生长、抗病性和过敏反应。
2水稻的两个基因OsLS和osRS的功能
本研究首次克隆到分别催化水稻核黄素合成过程的倒数第二步和倒数第一步的关键酶的编码基因OsLS和OsRS。OsLS基因全长666 bp，在氨基酸序列上与拟南芥(Arabidopsis thaliana)、菠菜(Spinacia oleracea)和烟草(Nicotiana tabacum)的LS基因的同源性非常高，在68-76％之间，根据氨基酸序列分析绘制系统进化树，在同一个进化分枝内。与稻瘟病菌(Magnaporthe grisea)和发光细菌(Photobacteriumphosphoreum)的核苷酸序列完全没有同源性，氨基酸序列有一定的同源性，分别是33％和48％，根据氨基酸序列分析绘制系统进化树，在不同的进化分枝内。在大肠杆菌内能表达出包括His-tag在内的约29．85 kDa大小的OsLS蛋白质。该蛋白质有221个aa，分子量是22，471．44 Da，蛋白质等电点是10．01，是一种碱性蛋白质。远远高于激发子HpaG_(xoo)蛋白质溶液的浓度(100μg／ml)的OsLS蛋白质水溶液注射烟草叶片不能引起过敏反应，这说明该蛋白质本身对植物的细胞没有损害。软件分析该蛋白质定位在叶绿体内，与拟南芥、菠菜和烟草的LS蛋白质的定位相同，OsLS蛋白质单体有5个α螺旋，4个β折叠，5个α螺旋把4个β折叠包围在中间。OsRS序列全长1662 bp，与Gene ID OJ1111H02．Predgene04的不含内含子和非翻译区的序列(no intronsequence and no other untranslated regions)完全一致。NCBI Blast分析，OsRS基因与A．thaliana(拟南芥基因库内的推测序列)、Candida albicans、C．famata、Filobasidiellaneoformans、Sinorhizobium meliloti、Bartonella elizabethae、Saccharomyces cerevisiae、Pichia guilliermondii、P．Phosphoreum、Schizosaccharomyces pombe、E．coli等的RS基因在核苷酸序列和氨基酸序列上都没有同源性，在系统进化树的不同进化分枝内。在大肠杆菌内表达该蛋白质，能表达包括His-tag在内的约66．37 kDa大小的蛋白质。100μg／ml的OsRS蛋白质水溶液注射烟草叶片不能引起过敏反应，这说明OsRS蛋白质本身对植物的细胞没有损害。软件分析该蛋白质有553个氨基酸，分子量是59，589．80 Da，等电点是6．00，酸性蛋白质，定位在叶绿体内，该蛋白质的折叠结构有6个α螺旋，5个β折叠，6个α螺旋把5个β折叠作为核心包围在中间。
OsLS和OsRS转基因烟草的游离态的核黄素、FAD和FMN含量都有所升高，而且OsLS转基因的烟草(LST)核黄素含量略高于OsRS转基因烟草(RST)的核黄素含量。LST和RST比转空载体的烟草对照植株(VECT，CK)营养生长旺盛，幼苗期表现更强烈，能使细胞壁疏松、促进植物生长的expansin genes在LST和RST内表达水平比CK表达水平高。LST和RST与对照相比细胞编程死亡和细胞死亡的标志基因hinl和hsr203的表达没有显著差异。与对照相比LST和RST抗病性增强，而且LST的抗病性强于RST。在接种TMV之前，LST和RST的抗病相关基因PR1a、PR1b的表达量都远远高于对照烟草内的表达量，接种TMV之后的同样时间，LST和RST内的PR1a、PR1b都受到诱导表达，受诱导的量远远大于对照烟草内受诱导的量，而且LST内PR1a和PR1b的整体表达水平高于RST内的表达水平。乙烯是重要的介导生长和防卫反应的信号分子，释放量增多能引起植物的生长加快和防卫反应增强。LST和RST比对照烟草乙烯的产生速度快、产生量大，RST在开始的乙烯产生量高于LST，但是LST随着时间变化增长幅度比RST大，LST和RST的生长和抗病的不同表现可能与这种乙烯的释放量有关。活性氧是一种与植物的编程性细胞死亡和抗病性密切相关的信号因子，低浓度的活性氧诱导植物的细胞死亡和抗病性，但是高浓度的活性氧会破坏植物的细胞膜，使植物丧失对病害的抗性。转基因的烟草在不接种病毒的时候，活性氧的产生量与对照烟草活性氧的产生量没有差异，接种病毒后48小时差异增大，对照烟草受到的活性氧的胁迫大于LST和RSt，而且LST活性氧产生量略小于RST，这种消除活性氧的胁迫的差异应该与抗病性的差异有关。
构建了OsLS和OsRS的过表达载体和沉默载体，转化水稻幼胚愈伤组织，成功得到OsLS和OsRS过表达的水稻转基因株系，OsgS部分沉默的水稻株系。OsLS发生过表达、完全或部分沉默很可能都对水稻的分化和生长产生严重的影响，转基因过程中发现，OsLS和OsLS片段的发夹结构转化水稻，愈伤组织开始分化比空载体转化的愈伤组织分化迟，但是OsLS片段的发夹结构转化的愈伤组织开始出现绿色后不能继续分化，逐渐褐化死亡。OsLS发生沉默可能是一个致死的过程，这与前人的研究细菌和真菌无法产生LS的突变体在一定程度上是一致的。OsRS转化的愈伤组织分化早，成苗快，OsRS片段的发夹结构转化的愈伤组织分化比空载体转化的愈伤组织分化迟，比OsLS转化的愈伤组织分化早。4个月苗龄的OsRS和OsLS过表达的水稻的游离态核黄素、FAD、FMN都比对照植株转化空载体的水稻(VECR)高；OsRS发生部分沉默的植株(SiRS)与VECR相比核黄素含量降低。OsRS转基因水稻(RSR)与VECR相比，分蘖多、长势好，控制分蘗的基因OsMOC1和生长活跃的标志基因OsGRF1表达量大大高于对照水稻内的表达量，抽穗时间大大提前。SiRS与VECR相比，分蘗较少、长势稍差，OsMOC1和OsGRF1表达量略低于对照水稻内的表达量，抽穗时间稍推迟。OsLS过表达株系(LSR)与对照相比，其分蘗能力和生长势大大下降，OsMOC1和OsGRF1表达量少于对照水稻内的表达量，抽穗时间大大延迟。三种株系的细胞死亡没有明显差异，也即OsLS和OsRS不影响植株的衰老。接种白叶枯病菌PX099，LSR对白叶枯病的抗性最好，病程相关基因OsPR1b和OsPR10受到强烈的诱导，RSR的抗性有小幅提高，OsPR1b和OsPR10受到较强烈的诱导，弱于LSR；SiRS的抗性明显降低，抗病性几乎完全丧失，OsPR1b和OsPR10几乎不受到诱导表达。与对照水稻相比，LSR和RSR受到的活性氧的胁迫减小，而且OsLS的抑制作用强于OsRS，但是SiRS受到的氧胁迫大于对照植株，较低的合适浓度的活性氧对植物的抗病性是有利的，但是过高浓度的活性氧会大大损害植物细胞。
总之，水稻的OsLS和OsRS具有非常重要的作用，分别对抗病和生长有很强的促进作用，都能消除活性氧的胁迫，而且OsLS的消除作用强于OsRS。这两个基因对植物的编程性细胞死亡都没有影响。OsLS在烟草和水稻上对生长的作用不同的原因值得深入研究。这两个基因在水稻的育种中有很重要的应用前景。
3烟草NtTRXh基因负调控营养生长，正调控抗病性，与细胞死亡无关
克隆到烟草(Nicotiana tabacum)的thioredoxin h-like protein complete CDS序列，该蛋白具有完整的TRX蛋白质的活性位点WCGPC。序列的同源性和系统进化树分析可见，NtTRX-h1(AF435818)与小麦的Tal(AF438359)、大麦的Hvh1(AF435815)和拟南芥的Ath-t1(AAG51342)中的氨基酸序列同源性非常高，分别达到71％、72％和78％，在同一个进化簇内；但是与番茄(Lycopersicon esculentum)的LeCITRX-pt(AF261142)和Solanum tuberosum的StCDSP32(Y09987)的氨基酸序列同源性很低，分别为30％和24％，在不同的进化簇内：与烟草(N．alata)的Nah(DQ021448)的氨基酸序列同源性也不高，为37％，在不同的进化簇内。在大肠杆菌内表达该蛋白质，能表达包括His-tag(大约5，426 Da)在内的约22，260 Da大小的蛋白质。150μg／ml的TRX蛋白水溶液不能引起烟草叶片的过敏反应，这说明该蛋白质本身对植物的细胞没有损害。软件分析该蛋白质有152个aa，分子量是17，022．20(17．02 kDa)，蛋白质等电点(pI)是4．53：很可能是一种线粒体基质空间内和细胞质内的蛋白质，微粒体和线粒体内膜上可能少量存在(或不存在)；TRX蛋白的高级构象有2个α螺旋，5个β折叠。
成功构建该基因到病毒沉默植物载体pBinPlus2β：：1．7A上，得到pBinPlus2β：：1．7A：：trx，转化农杆菌EHA105，注射烟草，部分沉默ZRX基因的转录表达。成功构建该基因到含有激发子诱导性启动子的载体pBI121：：PPP1上，得到pBI121：：PPP1：：TRX，转化农杆菌EHA105，叶盘法转化烟草，产生转基因的烟草PPP1：：TRX，以空载体pBI121：：PPP1转化烟草做对照。使用激发子harpin_(Ea)诱导转基因烟草发现，PPP1在受到harpin_(Ea)诱导后，能成功引起烟草内源基因TRX的过表达。TRX基因发生部分沉默的植株(siTRX)营养生长速度都比对照植株快，达到显著性差异，NtEXP1、NtEXP2和NtEXP6基因表达量增长；相反TRX基因发生过表达的植株(OvXRX)营养生长速度都比对照植株(CK)慢，达到显著性差异，NtEXP1、NtEXP2和NtEXP6基因表达量略有下降。SiTRX、OvTRX分别和对照植株相比细胞死亡没有明显差异，细胞死亡标志基因hin1和hsr203的表达水平也没有明显差异。植株的TRX基因发生部分沉默对FMV的抗性明显降低，病斑数大大增多，感病程度升高，达到显著性差异，抗病防卫反应受到严重损害，抗病相关基因PR1a和PR1b在TRX发生沉默但是没有病毒侵染的情况下，表达量高于非沉默植株，这对植物本身不利，造成内源物质的浪费，但是一旦发生TMV病毒侵染，非沉默植株的基因表达量在24小时后迅速高于沉默植株，也就是TRX基因可能干扰了PR基因的诱导表达，相反植株TRX基因发生过表达对TMV的抗性明显升高，病斑数大大减少，抗病性增强，达到显著性差异，抗病性增强，抗病相关基因PR1a和PR1b在TRX发生过表达时一旦发生TMV病毒侵染，OvTRX的基因表达量在24小时后迅速高于对照植株，也就是TRX基因可能增强了PR基因的表达。活性氧与植物的抗病性密切相关，高水平的活性氧产生会使植物迅速丧失抗病能力。接种TMV后，SiTRX和OvTRX活性氧的产生在前24小时与对照植株都没有差异，但是从第48小时，SiTRX的活性氧产生量大大多于CK，大量的活性氧损害了PR基因的诱导表达，破坏了烟草的抗病机制，相反OvTRX的活性氧产生量大大低于CK的产生量，植株受到的氧胁迫降低，抗病性增强。总之，TRX基因抑制烟草的营养生长，诱导抗病性，不影响编程性细胞死亡。全文总结
通过上述研究，我们对激发子HpaG_(xoo)与生防细菌和ParA1的互作、水稻核黄素合成过程的酶2，4-二氢喋啶合酶和核黄素合酶、烟草的硫氧还蛋白对植物的生长发育和抗病性的作用有了初步的认识。首先，激发子HpaG_(xoo)能与生防细菌P．cepacia、B．subtilis和激发子ParA1互作，影响植物的生长、抗病性和过敏反应。其次，水稻的OsLS和OsRS具有非常重要的作用，OsLS对抗病性有很强的诱导作用，OsRS对生长具有很强的促进作用，二者都能消除活性氧的胁迫，而且OsLS的消除作用更强烈，这两个基因对植物的编程性细胞死亡都没有影响。这两个基因在水稻的育种中有很重要的应用前景，有利于培育生长周期短、产量高、抗病性好的品种。最后，TRX基因抑制烟草生长，诱导抗病性，不影响编程性细胞死亡。
本研究为开发更优化的病害防治策略，培育具有优良生长和抗病性状的品种，研究核黄素信号途径与氧化还原信号在植物的生长和抗病性上的信号网络和目标蛋白的研究准备了坚实的理论基础和转基因材料的物质基础，下一个要解决的问题是寻找这些基因在植物内的目标蛋白和相交叉的信号通路，探索这些通路之间的互作网络。
Due to public concerns on pesticide use in crops, exploration on alternative methods has been a global effort to secure crop. Biocontrol bacteria and natural products from various sources show a great potential of agricultural use in disease control and crop product improvement. Pseudomonas and Bacillus species is an important class of bioeontrol bacteria; and they protect plants with many mechanisms. Moreover, biocontrol bacteria can promote seed germination and plant growth with indirectly increasing disease resistance. Plant responses are induced by the biocontrol bacteria colonization on plant roots and play a role in disease control depending on the response speed and magnitude in contrast to the infection to plants by pathogens. Natural products which have a potential in crop improvement are various in nature and sources. HpaG_(Xoo), a harpin produced by Xanthomonas oryzae pv. oryzae, stimulates plant growth and resistance against pathogens and insects. Moreover, the biocontrol bacteria colonization on plant roots activates induced systemic resistance(ISR), which antagonizes SAR and provides plants with a distinct battery of defense arsenal. Therefore, a combinative use of biocontrol bacteria and the type-Ⅲeffectors could be more effective than use of either of them in crop improvement. Previously, we used P. cepacia P6854 and B. subtilis B-916 to control rice sheath blight with desired results. We have introduced HpaG_(Xoo) into rice(variety R109), generating several transgenic lines. They were improved in disease resistance. Here we show that the HpaG_(Xoo)-expressing rice line 1(HER1) increases growth and activates defenses toward salinity and pathogens. We present evidence that P. cepacia P6854 and B. subtilis differentially affects growth and disease resistance in R109 and HER1.
Elicitins are a peculiar of proteins produced by oomycetes in the genera Phytophthora and Pythium, and ParA1 is 10 kDa elicitor produced by P. parasitica var. nicotinanae, host special elicitor, sensitive to heat and protease K in vitro. Elicitins induce not only plant hypersensitive response(HR) but also defense reponses. In order to get more effective and more economical recombinant bacteria than the recombinant E. coli BL21(DE3) that expressing ParA1 and HpaG_(Xoo) singly, we constructed a bacterium expressing HpaG_(Xoo) and ParA1 simultaneously. The protein of the bielicitor bacterium has been tested for its effect on tobacco micro-HR and the resistance against tobacco mosaic virus.
Riboflavin is an important ingredient of flavin mononucleotide(FMN) and flavin adenine dinucleotide(FAD) in bacteria and eukaryotic organisms. FMN and FAD participate in many enzyme catalizing reactions, so riboflavin is a multifunctional vitamin which is essential for the maintenance of life. The two enzymes, lumazine synthase and riboflavin synthase, catalyze the last two steps in the biosynthesis of riboflavin. The two enzymes represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with the enzymes of the mammalian metabolism. The genes of many bacteria and yeasts have been cloned, but there are only little reports on plants and no research on rice. It has nearly been reported that the cos1 mutation(LS mutation) restores the coil-related phenotypes, including defects in JA sensitivity, senescence and plant defense responses in Arabidopsis, but there have no research about the functions on growth, disease resistance, active oxygen and hypersensitive cell death of the two genes of rice by so far. It is worth to research.
Thioredoxins are small proteins catalyzing thiol-disulfide interchange and are involved in the regulation of the redox environment of the cells. The plant thioredoxin system is particularly complex since many thioredoxin isoforms are found in plants and they are multifunctional proteins. Based upon primary sequence analysis and subcellular localization, thioredoxins can be classified into different groups and subgroups. The functions of tobacco thioredoxin h-like protein have not been reported, we try to research this gene actions on plant growth and disease resistance.
This study is focused on crosstalk between representative and distinct agencies that induce plant defense and growth, and functions of pivotal regulators in redox-based signal transduction that regulates both effects in plants. The interactions of HpaG_(Xoo) with P. cepacia or ParA1, and functions of the genes encoding lumazine synthase and riboflavin synthase from rice, and a gene encoding h-like thioredoxin from tobacco, as well, have been studied by multiple methods.
1 Bioelicitor and PGPR interact to affect plant growth and disease resistance Expression of HpaG_(Xoo), a bacterial type-Ⅲeffector, in transgenic plants induces disease resistance. Resistance also can be elicited by biocontrol bacteria. In both cases, plant growth is often promoted. Here we address whether biocontrol bacteria and HpaG_(Xoo) can act together to provide better results in crop improvement. We studied effects of P. cepacia and B. subtilis on the rice variety R109 and the hpaG_(Xoo)-expressing rice line HER1. Compared to R109, HER1 increased growth, grain yield and defense responses toward diseases and salinity stress. Colonization of roots by P. cepacia or B. subtilis caused some increase, in contrast to controls, in root growth of R109. Growth of R109 leaves and stems and HER1 roots were effected a little but leaves and stems of HER1 was inhibited. When P. cepacia and B. subtilis colonization was subsequent to plant inoculation with Rhizoctonia solani, a pathogen that causes sheath blight, the disease was less severe than controls in both R109 and HER1; HER1, nevertheless, was more resistant, suggesting that P. cepacia or B. subtilis and HpaG_(Xoo) cooperate in inducing disease resistance. Several genes that critically regulate growth and defense behaved differentially in HER1 and R109 while responding to P. cepacia or B. subtilis. In R109 roots, the OsARF1 gene, which regulates plant growth, was expressed in consistence with growth promotion by P. cepacia or B. subtilis. Inversely, OsARF1 expression was coincident with inhibition in growth of HER1 leaves. In both plants, the expression of OsEXP1, which encodes an expansin protein involved in plant growth, was concomitant with growth in leaves and roots, in response to P. cepacia or B. subtilis. We also studied OsMAPK, a gene that encodes a mitogen-activated protein kinase and controls defense responses toward salinity and infection by pathogens in rice. In response to P. cepacia or B. subtilis., an early expression of OsMAPK was coincident with R109 resistance to the disease, while HER1 expressed the gene similarly whether P. cepacia was present or not. Evidently, P. cepacia or B. subtilis and HER1 interact differently in rice growth and resistance. Whereas combinative effects of P. cepacia or B. subtilis and HpaG_(Xoo) in disease resistance have a great potential in agricultural use, it is interesting to study mechanisms that underlie interactions involving biocontrol bacteria, type-Ⅲeffectors and pathogens.
HpaG_(Xoo) and ParA1 are two important elicitors produced by plant pathogens, the genes parA1 and hpaG_(Xoo) were cloned by polymerase chain reaction and ligased into the expressing vector pET30a(+), transformed into E. coli BL21(DE3). The protein of BL21:: parA1:: hpaG_(Xoo) (ParA1:: HpaG_(Xoo)) was extracted, and electrophoresed by Tris-Tricine SDS-PAGE, 15 kDa and 10 kDa two proteins were got. The ParA1 and HpaG_(Xoo) were identified expressing synchronously because of different macroscopic hypersensitive response(HR) of tobacco leaves injected with the proteins boiled 30 min or not. ParA1:: HpaG_(Xoo) induced stronger microscopic hypersensitive response(micro-HR), systemic acquired resistance(SAR) to tobacco mosaic virus and expression of pathogen related genes, PR1a and PR1b than ParA1 or HpaG_(Xoo) only did.
2 The roles of lumazine synthase and riboflavin synthase in plant growth and defense
Based on transgenic and molecular studies, the functions of both enzymes from rice were researched in this study.
OsLS and OsRS are first cloned. OsLS have 666 nucleotides. The nucleotide sequence identities of LS genes of Oryza sativa, Arabidopsis thaliana, Spinacia oleracea and Nicotiana tabacum are between 68-76％, and the genes are in the same branch of phylogenetic tree. The nucleotide sequences of LS genes of Magnaporthe grisea and Photobacterium phosphoreum have no identity, but the amid acid sequences of them have 33％and 48％identities and the genes are in different branches of phylogenetic tree. OsLS protein with His-tag(29.85 kDa) was expressed in E. coli. OsLS protein has 221 aa, its molecular weight is 22, 471.44 Da, and pI is 10.01.100μg/ml OsLS protein aqueous solution cann't induce tobacco hypersensitive response, meaning this protein is not bad for plants. The protein, analyzed online, is localized in chloroplast, as the same as the location of the LS proteins of A. thaliana, spinach and tobacco. Its tertiary structure has 4μfolds, surrounded by 5αhelixes. OsRS has 1662 nucleotides, having 100％identity with Gene ID OJ1111H02. Predgene04. NCBI Blast analyed that OsRS has no identity with the RS sequences of A. thaliana, Candida albicans, C. famata, Filobasidiella neoformans, Sinorhizobium meliloti, Bartonella elizabethae, Saccharomyces cerevisiae, Pichia guilliermondii, P. phosphoreum, Schizosaccharomyces pombe and E. coli, and the genes are in different branches of phylogenetic tree. OsRS protein with His-tag was expressed in E. coli, whose molecular weight is 66.37 kDa. 100μg/ml OsRS protein aqueous solution can't induce tobacco hypersensitive response, meaning this protein is not bad for plant. It has 553 aa, the molecular weight is 59, 589.80 Da and pI is 6.00. The protein analyzed online is localized in chloroplast, and its tertiary structure has 5βfolds, surrounded by 6αhelixes.
OsLS and OsRS trangenic tobacco have higher levels of free riboflavin, FMN and FAD than that of the blank vector transgenic tobacco(VECT), and the OsLS transgenic tobacco (LST) have higher level than OsRS transgenic ones(RST). LST and RST have better growth in infancy and youthful times than the wild type(WT) and VECT. Expansin genes, which enloose cell wall and enhance plant growth, of LST and RST have higher expression levels than VECT and WT. RST growed better than LST. OsLS and OsRS have no effect on programmed cell death in transgenic tobacco lines compared with WT and VECT, the marker genes hin1 and hsr203 have similar expression levels in them. The disease resistances of LST and RST were enhanced, the pathogenesis-related genes(PR) were induced stronger than control(CK), and LST have better resistance than RST. Ethylene is an important signal molecular related to development and disease resistance. LST and RST can produce more ethylene than CK, this may resulted into fast growth and strong disease resistance. Active oxygen is important for plant disease resistance, and the active oxygen of high level destroy plant disease resistance. Without TMV inoculation, LST and RST have no difference with CK, but LST and RST were haunted by less active oxygen stress, and especially LST has the least one, this means OsLS has sronger energy to eliminate active oxygen stress than OsRS.
Rice immature embryo calli were transformed with the overexpressing vector and hairpin unit of OsLS and OsRS by soaking the plant material with an appropriate Agrobacterria tumefaciens EHA105 suspension. OsLS and OsRS overexpressing rice(LSR and RSR) and OaRS partly silencing lines(SiRS) have been got, but OsLS haipin transformed rice(SiLS) was not got. The rice calli transformed by OsLS hairpin rice couldn't differentiate. The calli transformed by OsLS overexpressing vector rice grew and differentiated slower than those transformed by the blank vector, but the rice calli transformed by OsRS overexpressing vector grew and differentiated fast. The rice calli transformed by OsRS hairpin vector grew and differentiated slower than CK. Four-month old LSR and RSR have higher levels of free riboflavin, FAD and FMN than CK. SiRS have lower levels of riboflavin, FAD and FMN than CK. RSR have more tillers than CK, but LSR and SiRS have fewer tillers than CK. OsMOC1, which controls tiller formation and OsGRF1, which expresses in actively growing parts, have higher expression levels in RSR than CK, but have lower expression in LSR and SiRS than CK. All lines have no marked difference in programmed cell death. The disease resistance of LSR was enhanced the most among RSR, VECR, SiRS and LSR. RSR were enhanced a little disease resistance than VECR. The defenee response of SiRS were damaged absolutely. The expression levels of pathogenesis-related genes, OsPR1b and OsPR10, were in the same trend with rice blight resistance. LSR and RSR were effected by less active oxygen stress than VECR, but SiRS haunted by more active oxygen stress than CK. So OsLS and OsRS are very important, OsLS induces disease resistance and inhibits growth, but OsRS induces growth and disease resistance, they all eliminate active oxygen stress.
3 Tobacco thioredoxin signaling inhibits plant growth but induces disease resistance and does not affect programmed cell death
Nicotiana tabacum thioredoxin h-like protein complete CDS sequence was cloned, with the conserved active domain WCGPC. The amino acid sequence identities of TRX with those of N. tabacum(NtTRX-hl, AF435818), Triticum aestivum(Tal, AF438359), Hordeum vulgare(Hvhl, AF435815) and Arabidopsis thaliana(Ath-tl, AAG51342) are 71％, 72％and 78％respectively, and the genes are in the same branch of phylogenetic tree. The identities with those of Lycopersicon esculentum(LeCITRX-pt, AF261142), Solanum tuberosum(StCDSP32, Y09987) and N. alata(Nah, DQ021448) are only 30％, 24％and 37％respectively, and the genes are localized in the different branches of phylogenetic tree. TRX protein was expressed with His-tag(5, 426 Da) in vitro, the molecular weight is 22, 260 Da. 150μg/ml TRX protein couldn't induce tobacco hypersensitive response, meaning the protein is not bad to plant. TRX protein has 152 aa, its molecular weight is 17, 022.20 Da, and its pI is 4.53. This protein is most possible in mitochondrial matrix space, and may be in cytoplasm, microbody(peroxisome) or mitochondrial inner membrane. TRX tertiary structure has 2α-helixes and 5β-folds.
TRX partially silenced tobacco plants(SiTRX) grew faster than the control(CK), the difference is marked, and the expansin genes, which enloose cell wall and enhance plant growth, NtEXP1, NtEXP2 and NtEXP6 have higher expression levels than CK. TRX overexpressing plants(OvTRX) have the reverse results compared with SiTRX. TRX silence and overexpression have no effect on hypersensitive cell death compared with CK, the marker genes hin1 and hsr203 had similar expression levels in LST, RST and CK. The disease resistance of SiTRX was damaged, the pathogenesis-related genes PR1a and PR1b were induced less than CK, and this means TRX disturbs these genes expression, in the other side OvTRX have better TMV resistance and higher pathogenesis-related gene expression levels than CK. Without TMV inoculation and within 48 inoculation, active oxygen levels were similar in SiTRX, OvTRX and CK, but 48 hours postinoculation SiTRX produced more active oxygen than CK, but OvTRX produce less than CK. Clearly, the TRX gene inhibits plant growth but induces disease resistance and doesn't affect hypersensitive cell death.
Conclusion
In a conclusion, we have some results about the interaction of HpaG_(Xoo) with P. cepacia, B. subtilis or ParA1, the functions of lumazine synthase and riboflavin synthase from rice and thioredoxin from tobacco. First, there are some interactions between HpaG_(Xoo) and P. cepacia, B. subtilis. or ParA1, which effected on plant disease resistance and hypersensitive response. Second, OsLS and OsRS have important roles on plant growth and disease resistance. OsRS enhances growth, OsLS strongly induces disease resistance, and the two genes doesn't affect programmed cell death. The two genes have important potentials on the culture of the varieties of short-time cycle, high yield and strong disease resistance. Finally, TRX inhibits tobacco growth, induces disease resistance and doesn't affect hypersensitive cell death.
The theory bases and transgenic plants are prepared to explore better plant protection strategy, and to culture the varities with better growth and disease resistance than CK, to study the roles of riboflavin pathway and redox signals in plant disease resistance and growth, and to look for their target proteins.
引文
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