【作者】 贾承国；

【导师】 汪俏梅；

【作者基本信息】 浙江大学 ， 蔬菜学， 2009， 博士

【摘要】 番茄（Solanum lycopersicum）是最重要的蔬菜作物之一,随着番茄遗传学研究和国际茄科基因组计划（International Solanaceae Genomics Project,SOL）的发展,番茄已成为研究植物激素相互作用的理想模式植物。茉莉酸（jasmonic acid,JA）作为一种新型的植物内源激素,既是植物生长发育的调节因子,又在植物对逆境的适应性中发挥重要作用,因而受到研究者广泛的关注。本文以转基因番茄和激素相关突变体为试材,研究了番茄中系统素/茉莉酸（systemin/JA）与油菜素甾醇类（Brassinostsroids,BRs）信号转导途径的相互作用,并研究了JA与其他激素途径在茎枯病抗性以及AAL-toxin诱导的细胞程序化死亡（prograrnmedcell death,PCD）中的作用。所取得的主要结果如下:1、建立了番茄转基因体系,将拟南芥中BR信号转导组分基因bzr1和bes1转入番茄中。通过PCR检测共获得5株bzr1和3株bes1阳性植株,RT-PCR分析表明,阳性植株中均有目的基因的表达。转基因植株均能正常生长结实,对获得的转基因后代进行遗传特性分析,结果表明3个bzr1转基因株系和1个bes1转基因株系中Kan抗性植株与敏感植株分离比符合3:1,表明这3个bzr1株系是单拷贝插入,1个bes1株系是单拷贝。2、以bzr1转基因植株为材料,对BR反应进行了分析。结果显示,在光照和黑暗条件下,BR处理转基因植株后下胚轴和根长度的变化与野生型变化一致,但是转基因植株中提高了BR生物合成基因Dwarf以及BR调节基因LeBR1的表达。MeJA处理dx,bzr1转基因植株以及野生型后,JA响应基因PI-Ⅱ的表达变化一致。与对照中蔬四号相比,bzr1转基因植株中JA生物合成基因（LOXD,OPR3和AOS2）的表达也没有明显差异。光照条件下,不同浓度的BL处理systemin/JA突变体（spr1,spr2和def1）及其野生型后,其根长变化趋势一致。黑暗条件下,BL及其抑制剂BRZ处理systemin/JA突变体（spr1,spr2和def1）后,其根和下胚轴长度变化与野生型相比没有明显差异。10^-7M的BL处理systemin/JA突变体后分析了BR调节基因Dwarf和LeBR1的表达,结果显示,与野生型相比,突变体中BR调节基因表达变化一致。以上结果表明,systenmin/JA伤信号与BR被膜受体感知后,在细胞质中的转导途径之间可能没有相互作用,而是拥有完全不同的下游组分来调节基因的表达和特定的生理反应。3、利用JA突变体（spr2,def1和jai1）,研究了JA生物合成和信号转导途径在番茄对链格孢菌（Alternaria alternata f.sp.lycopersici,AAL）引起的茎枯病的抗性中的作用机制。结果表明,JA能够提高番茄对茎枯病的抗性,但JA受体COI1对AAL感病是必需的。利用qRT-PCR分析了AAL真菌侵染jai1及其野生型后JA生物合成酶基因（LOXD和AOS2）以及水杨酸（Salicylic acid,SA）生物合成（PAL）和信号转导途径基因（LeNPR1和PR基因）的表达,结果表明:jail提高对AAL的抗性并不是由于提高了JA含量,也不是由于增强了SA的生物合成和信号转导途径。利用乙烯抑制剂1-MCP和乙烯过量产生突变体Epi,分析了乙烯在茎枯病抗性中的作用,结果表明乙烯提高了对AAL真菌的敏感性。利用qRT-PCR分析了AAL侵染jail及野生型后乙烯生物合成（LeACO1和LeACS4）及信号转导途径中一些基因（LeETR1,LeETR3和ERF1）的表达,结果显示:与野生型相比,AAL真菌侵染jail后明显抑制了乙烯生物合成和信号转导途径。在以上研究基础上提出了JA、SA及乙烯“对话”（cross-talk）在番茄对链格孢菌抗性中的作用的模式图。4、利用JA突变体（spr2,def1和jai1）,研究了JA生物合成和信号转导途径在AAL-toxin诱导的细胞程序化死亡中的作用。结果表明,JA缺失突变体（spr2和defl）增强了AAL-toxin诱导的PCD,而JA非敏感突变体jail降低了AAL-toxin诱导的PCD。研究了SA和乙烯在AAL-toxin诱导的PCD中的作用,结果显示,SA抑制而乙烯促进AAL-toxin诱导的PCD。基因表达结果表明,jai1降低PCD并非是通过提高SA途径来实现的,可能与乙烯反应以及过氧化氢的代谢有关。更多还原

【Abstract】 Tomato（Solarium lycopersicum）is one of the most important vegetable crops in the world.It has also become a good model system to study cross-talk of different plant hormones with the development of genetics and whole tomato genome sequencing from International Solanaceae Genomics Project（SOL）.Jasmonic acid （JA）is a fatty acid-derived signal and exists in the plant kingdom extensively. Because of its important roles in plant growth,development and defense response against stress,this signal molecule has aroused researchers’ great interest.In this study,we use transgenic plants and plant hormone-related mutants in tomato to investigate the cross-talk between systemin/JA and BR（Brassinosteroid）signaling pathways.Furthermore,experiments were also carried out to examine the cross-talk between JA and other plant hormones in defense response against AAL and AAL-toxin-induced PCD in tomato.The results are as follows:We first established an efficient tomato genetic transformation system mediated by Agrobacterium tumefaciens,and transformed bzr1 and bes1 genes which involved in BR signal transduction into tomato（cv.ZhongShuSiHao）.Five bzr1 and three bes1 transgenic tomato plants were obtained and verified by PCR detection.RT-PCR analysis showed that both genes were expressed in these plants,although the transcript levels varied among different transgenic lines.All transgenic lines grew normally at all development stages.We then analyzed the segregation ratio of T₀ seeds on 1/2 MS medium containing 200 mg/L Kan,and the results showed that the Kan-resistant and susceptible ratio of three bzr1 lines and one bes1 line were close to 3:1,which indicated that these transgenic lines were inserted as single copy according to Mendelian segregation rule.BR responses were analyzed in bzr1 transgenic tomato plants,the result showed that the effect of BL treatment on hypocotyls and root length was similar between bzr1 transgenic lines and ZhongShuSiHao under either light or dark conditions,but BR-biosynthetic Dwarf and BR-regulated LeBR1 genes were up-regulated in bzr1 transgenic tomato plants.The expression of JA-responsive PⅠ-Ⅱgene in dx,bzr1-3 （transgenic line）and ZhongShuSiHao was similar at different periods after MeJA treatment.The transcript levels of JA biosynthetic genes（LOXD,OPR3 and AOC） were similar between bzr1-3 and ZhongShuSiHao.The root length of systemin/JA mutants（spr1,spr2 and def1）were inhibited as well as WT after BL treatment under light.In the dark,root and hypocotyl length of systemin/JA mutants were not significantly different with those of WT after BL or BRZ treatment.Furthermore,no significant difference in Dwarf and LeBR1 expression after BL treatment was observed among systemin/JA mutants and WT.These results indicated that unlike the Toll signaling in animal cells,the systemin and BR response in tomato did not share any common components downstream of the receptor,but own the distinct downstream components to regulate specific gene expression and physiological response.The role of JA biosynthesis and signaling pathway in resistance or susceptibility to toxin-producing fungal AAL was investigated by utilizing several JA mutants. Tomato mutants with reduced JA levels（def1 and spr2）were much more susceptible to AAL than WT.However,another JA-insensitive mutant jai1 showed higher resistance to AAL than the related wild type.These results indicated that the octadecanoid signaling pathway was required for defense response while JA receptor COI1 was essential for susceptibility to this pathogen in tomato.The expression of JA-biosynthetic genes（LOXD and AOS2）and salicylic acid（SA）-regulated genes {LeNPR1 and PR)were reduced in jai1 compared with WT during the pathogen infection,indicating that jai1 enhanced resistance was not dependent on elevated JA accumulation or SA signaling pathway.Role of ethylene（ET）in tomato-AAL interactions was also investigated by using ethylene receptor inhibitor 1-MCP treatment and an ethylene overproduction mutant Epi.The results demonstrated that ET response was involved in susceptibility to AAL instead of defense response. Further data showed that genes involved in ET biosynthesis（LeACO1 and LeACS4） and signaling（LeETR1,LeETR3 and ERF1）were down-regulated in jai1 after AAL inoculation.Based on our studies,a model was proposed to summarize the role of different plant hormone signaling,including JA,SA and ethylene and their cross-talk in response to AAL.The role of JA biosynthesis and signaling pathway in AAL-toxin-induced PCD was also studied by using JA mutants.The results showed that JA-deficient mutants spr2 and def1 enhanced AAL-toxin-induced PCD,while JA-insensitive mutant jai1 inhibited.We also investigated the role of SA and ethylene in this process,and the results demonstrated that SA and ethylene had opposite effects on PCD,SA improved, while ethylene reduced AAL-toxin-induced PCD.Gene expression analysis by qRT-PCR revealed that jail decreased AAL-toxin-induced PCD was independent of SA signaling,but related to ethylene response and ROS metabolism.更多还原