【作者】 姚桢；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2004， 硕士

【摘要】 梨火疫病菌产生的harpin_Ea、水稻白叶枯病菌产生的harpin_Xoo和水稻细条斑产生的harpin_Xooc，作为广谱性激发子都可以诱导植物产生多种有益表型如抗病、抗虫、抗旱、促生长等。究竟harpins启动哪些信号通路来赋予植物这些表型?什么信号通路控制植物生长加快或抗旱?每个信号通路需要哪些调控因子?本研究立足于这些问题，解析harpins促进植物生长和诱导抗旱的信号传导机制。Harpin的作用位点与后续的信号传导息息相关，关于harpins在植物上的作用部位一直是研究热点，但究竟位于细胞壁、细胞膜还是细胞质中，针对不同harpin的研究有不同的结论。作者荧光蛋白标记了harpin_Xoo和harpin_Xooc试图解析它们在水稻中的作用部位。 1．Harpins促进植物生长信号通路解析 作者试验证明，harpin_Ea可以促进多种植物生长。Harpin_Ea处理植物拟南芥、番茄、烟草、水稻可明显促进植物根系、茎叶生长，提高叶片全氮含量、叶绿素总含量及叶绿素a／b，增强光合作用、提高光合速率。在拟南芥上，harpin_Ea处理可以诱导植物生长发育信号通路中关键基因PDF1.2、PR-3b、ERS1、ETR1和EIN2的表达。PDF1.2和PR-3b是乙烯信号通路的分子标志，而乙烯信号传导可以控制植物抗病、抗虫以及植物生长发育；ERS1、ETR1和EIN2是乙烯信号通路中上、下游的关键调控基因，其产物分别作为乙烯的受体和转录调控因子起作用。研究还发现，harpin_Ea可以促进一类伸展素基因（expansin）的表达，expansins在细胞的快速生长中起着重要的作用。根据以上结果，harpin_Ea启动了植物生长信号传导，促进了植物的生长。 2．Harpins诱导抗旱与ABA信号通路的关系 烟草用harpin_Xoo喷雾处理后，对人为断水干旱的抗性显著增强。人工干旱第7天时，harpin_Xoo处理的植株生长正常，与正常浇水和ABA处理的相似；而H₂O处理的对照植株中下部叶片已经萎蔫，继续干旱到第10天时大多数植株完全萎蔫或死亡。显微镜观察发现，harpin_Xoo处理的植株叶片上气孔孔径显著变小，干旱到第六天时，大约有70％的气孔已经完全关闭，而对照植株只有24％的气孔关闭或变小。Harpin_XooH呷in促进植物生长和诱导抗早的信号传导解析以及定位的初步研究还引起一些与杭旱有关的生理变化，在干旱胁迫下，h抑inxoo处理植株的细胞膜损伤程度明显低于对照植株，harpinx。。诱导植株体内脯氨酸和ABA含量急剧增加，减缓超氧化物歧化酶活性的降低及丙二醛积累。Rl’- PcR检验表明，h抑inxoo能诱导或增强ABA信号通路中关键基因osmotin、pkll一cI和phi一2的表达。据此，ABA可能参与harpinxoo诱导的植物过程。3.Harpinxo。与harpinxo。。荧光标记及作用定位的初步研究 已有研究试图确定harpins在植物细胞中的作用部位。但对不同的harpins如来自五洲，sniaa柳lovora的h抑inEa、Pseudomonas撰in卯e pv.phaseolieola的haprinPsph等研究结果都不尽相同.因此，h抑ins的识别部位、结合受体等一直不清楚。在本研究中，作者利用荧光蛋白标记，试图研究h抑inxoo和h呷inxooc在植物中的作用部位。Harpinxoo和harpinxooc分别由hrfi基因和h价基因编码。作者把hrf!和h榨基因分别与荧光蛋白加（green fluoreseent protein）/咖（red fluoreseent protein）构建融合基因，转化pE下3oa（+）载体，获得了重组质粒pET30::hrf7::肺、pET30::hrf7::rfP、pET30::h币::加和pET30::h币::rfP，并分别转化丑coli BLZI（D E3），产生了表达菌株pGH盯1、pRH盯1、pGHRFZ和pRH盯2。在IPTG诱导条件下培养转化菌株，收集的菌体经超声波破碎后均能在烟草叶片上引起典型的过敏反应，在SDS一PAGE电泳上出现相应大小的蛋白质条带.同时，把融合基因hrf]::加和h价::咖构建到载体pURF034中，利用517一l菌株通过两亲交配分别转化到水稻白叶枯小种JXOm和细条斑小种RS105中，接种水稻品种汕优63，试图通过荧光标记进行harpinxoo和h呷访xooc在水稻中的定位。4.总结 通过以上研究，我们得出三点结论.第一，harpins诱导乙烯信号通路中关键基因的表达，促进植物生长，增强光合作用。第二，h抑ins明显增强植物的抗旱性，促进气孔关闭，减少水分丧失。第三，荧光标记了harpinxoo和harpinxoo。，为后续的定位研究莫定了基础。但是本研究还存在一些缺点.第一，对h呷ins促进生长和抗旱信号通路的研究，只是作了较多的表型测定，缺乏更直接的证据来解析这些通路的重点环节及信号交又。第二，关于荧光定位两种h呷ins的研究，还无明确结论。因为，把荧光标记的菌株剪叶接种水稻品种汕优63以后，在荧光显微镜下，视野中发出明显的亮光，不能够进行细微观察和准确定位，用激光共聚焦显微镜观察可能会避免这些问题。第三，我们选用了带有荧光的质粒pUFR034标记菌株，有一定的缺陷。因摘要为质粒上的报告基因及杭生素基因的表达是在宿主菌体的复制、转录、翻译系统作用下进行的，容易消耗宿主的物质能量，而且连续培养条件下，质粒也很可能丢失。更多还原

【Abstract】 Harpins are glycine-rich, protease-sensitive, heat-stable, acidic proteins produced by Gram-negative plant pathogenic bacteria, and are required for induction of the hypersensitive response (HR) or hypersensitive cell death (HCD) in nonhost plants of bacteria. Application of harpins to many plants can enhance plant growth, induce resistance to pathogens or insects and improve tolerance to environmental adversity like drought.However,what signaling processes contribute to enhanced plant growth (EPG) or drought tolerance (DT) in plants treated with harpins have been unclear. This study was aimed to address questions on harpin signaling in EPG and DR by determining whether the phenotypes are attributable to activation of signaling pathways. By now, the target sites of harpins on plant cells are not clear.The cellular localization of harpins are closely related to the contituative signal transduction.However as which part of the plant cell is cell wall,cell membrance or cytoplasm ,there is no consistent conclusion from different research. The author tried to use the fluorescent protein labelled harpinxoo and harpinxooc to analyze their cellular localization in rice plant. 1. Harpin enhance plant growthHarpinEa can enhance plant growth in coincidence with relevant physiological responses and expression of several genes involved in ethylene signaling pathway.In the assay on four different kinds of plants treated with harpinEa including Arabidopsis, tomato, tobacoo and rice were taller and their individual weight were higher than those treated with water. Total leaf nitrogen content (TLN),chlorophyll a/b ratio(Chl.a:b) and chlorophyll content (Chi) which were connected closely with photosynthesis of plant were measured at seven days after treatment with harpinEa and CFVP(cell-free vector preparation).These results suggested that harpinEa can improve the TLN ,Chl and Chl.a:b. Application of to Arabidopsis thliana induces expression of the EIN2, ETR1, ERS1,PDF1.2, andPR-3b genes.Whereas PDF1.2,and PR-3b are molecular markers of the ethylene signal transduction pathway,which regulates growth and development and many other processes in plants, ERS1,ETR1 and EIN2 function as ethylene receptors and a transcriptional regulator in the pathway. Further,application of harphiEa induces expression of genes encoding expansin that played significant role in plant growth.2. Harpin -induced DT is mediated by ABA in tobacooHarpinxoo conspicuously increased drought tolerance (DT) of plants which water had been withheld for seven days before treatment with the protein and subsequently grown under water stress conditions for another 10 days. Up to 7 days of water stress (WS),Harpinxoo-treated plants grew well as those regularly watered. In contrast, control plants sprayed with water became wilt and faded away soon.Moreover, microscopic observation showed evident differences in stomatal status between plants treated water and Harpinxoo-By 6 days after the protein application and 7 days after WS, approximately 70% stomata closed. In contrast, during the same period, only 24% stomata closed on leaves of control plants.Harpinxoo effectively reduces some drought severity.Harpinxoo can also induce other physiological characteristics related to drought tolerance.These include: increasing the levels of endogenous proline, reducing cell membrane permeability . improving superoxid dismutase (SOD) activities and decreasing the content of malondialdehyde (MDA).In these responses, harpinx00 acted as similary as did ABA.However harpinx00 kept at lower levels than ABA-control,and no changes are found in the negative control in which the plants were treated regularly with water. In plant grown under water stress conditions,harpinxoo induced the accumulation of endogenous abscisic acid similarly to ABA. However the amount of increase and timing are different between two treatments. Compared to the control plants, faster and larger increase in endo-ABA concentration was found in plants treated with harpinxoo- Genes osmotin, pkll-cl andphi-2 were expre更多还原