【作者】 洪薇；

【导师】 曹家树； 郑重；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 博士

【摘要】 番茄(Lycopersicon esculentum Mill.)对叶霉病(Cladosporium fulvum)的抗性是番茄抗C.fulvum基因(Cf)和互补的C.fulvum无毒基因(Avr)产物进行识别互作,激活下游抗病信号传导,并活化各类防卫反应基因表达的结果。Cf-9与Cf-4蛋白的氨基酸序列91%以上一致,而Avr9与Avr4则没有明显的同源性,Avr9/Cf-9和Avr4/Cf-4介导的过敏性坏死(hypersensitive response,HR)在坏死产生时间、方式和强度上表现明显差异。Avr9/Cf-9番茄苗中HR产生早,扩展慢,产生的过敏性坏死斑小而多,分散,在叶脉上较少见。而Avr4/Cf-4苗中HR产生迟,扩展快,产生的过敏性坏死斑少,多产生于主脉,常沿叶脉迅速扩大成坏死区,最终导致整个子叶的萎蔫脱落。导致两种Avr/Cf苗产生不同HR的原因,可能是Cf表达的差异,和/或Cf下游信号传导途径的差异。本研究以通过杂交获得的携带Avr9/Cf-9或Avr4/Cf-4基因对的番茄为材料,观察降温过程中Avr9/Cf-9和Avr4/Cf-4番茄幼苗子叶HR坏死产生过程的异同;通过cDNA-AFLP分析,比较和分离克隆Cf-9和Cf-4下游信号传导相关基因(片段),并对分离到的部分相关基因进行功能分析,建立对cDNA-AFLP片段直接进行抗病相关性验证的TRV介导的VIGS分析体系;比较分析植株发育、温度以及互补无毒基因对Cf-9和Cf-4基因表达的影响;以便增进对Cf-9和Cf-4介导HR和抗病性产生的分子机理的理解,并为采用基于cDNA-AFLP片段的基因沉默技术快速大量分离鉴定植物抗病相关基因奠定基础。获得的主要研究结果如下:(1)对Avr/Cf番茄幼苗产生HR的特性进行显微观察和比较分析,发现高温33℃抑制Avr/Cf番茄中HR的产生,5 d苗龄的Avr9/Cf-9和Avr4/Cf-4幼苗从33℃降温到20℃培养后4h和16h有少许细胞被锥虫蓝染成蓝色,两者分别在降温12h和48h后大片组织被染成蓝色,说明Avr9/Cf-9和Avr4/Cf-4番茄苗分别于降温后4h和16h后开始产生HR,至12h和48h形成典型HR。(2)采用上述Avr/Cf番茄苗通过温控产生HR的系统,对分别为Avr4、Cf-4、Avr4/Cf-4、Avr9、Cf-9、Avr9/Cf-9的6个基因型番茄苗经高温和降温处理后所取样品进行cDNA-AFLP分析。通过512对选扩组合对12个样品共得到279 480个明显cDNA-AFLP条带,其中表现和不表现HR时差异表达的ACE(Avr/Cf-elicited,either induced or repressed)条带为367个。在这些差异表达条带中,249个为Avr4/Cf-4和Avr9/Cf-9番茄苗产生HR时均诱导表达,118个为产生HR时表达均受抑制。共克隆差异表达最显著的序列189个,均已登录GenBank核酸序列数据库,登录号分别为CD579099～CD579164,CK348286～CK348390,以及DN956215～DN956225。这些差异表达片段对应基因分别编码抗病蛋白,以及与HR/细胞坏死、信号传导、防卫反应、转录调节、代谢,和蛋白合成等相关因子。此外,共获得cDNA全长序列9个,因其皆为Avr/Cf诱导表达,故称为ACI(Avr/Cf-induced)基因,登录号分别为DQ056433～DQ056442。其中,ACI14编码钙调磷酸酶类磷酸酯酶(calcineurin-like phosphoesterase family protein),ACI25编码木葡聚糖特异性真菌内源葡聚糖酶抑制子蛋白(xyloglucan-specific fungal endoglucanase inhibitor protein),ACI29编码60S核糖体蛋白L10,ACI36编码酮戊二酸/苹果酸转运蛋白(oxoglutarate/malate translocator),ACI37编码细胞色素b559,ACI39编码胞质核糖体蛋白S13,而ACI13,ACI19和ACI112编码功能未知的蛋白。cDNA-AFLP差异显示结果表明,虽然367个ACE片段在Avr9/Cf-9和Avr4/Cf-4苗中表现与不表现HR时差异表达的趋势(即同样被HR诱导或抑制)一致,但其中42.8%(157/367)ACE片段在两类HR中差异表达的强度不同,绝大多数(135/157,86.0%)在Avr4/Cf-4苗中差异表达程度更显著。这些结果说明,Cf-4和Cf-9下游信号传导途径涉及的基因很可能一致,但它们表达的精细调控可能有一定区别。(3)对ACI29在HR产生前后和胁迫处理后的表达进行分析,发现在只含Avr9、Cf-9、Avr4或Cf-4基因不产生HR的番茄苗中,ACI29在苗龄为3d及以前的小苗中微弱表达,但在4d后表达明显增强。在有互补Avr/Cf基因对存在因而产生HR的幼苗中,ACI29表达比上述对照强,且Avr9/Cf-9和Avr4/Cf-4番茄苗之间,ACI29表达时序和强度都有明显差异,在Avr9/Cf-9幼苗中的表达早于Avr4/Cf-4幼苗,但在Avr4/Cf-4苗HR产生后,ACI29的表达比同样大小的Avr9╱Cf-9苗中更强烈。说明ACI29表达与植株的发育和过敏性坏死程度相关。另外,重金属离子处理微弱诱导ACI29的表达。盐渍和干旱强烈抑制其表达,而机械损伤处理后的表达与未处理的对照相当。这说明60S核糖体蛋白L10可能参与植物的发育、HR和逆境反应的调控。(4)分别将差异片段29和14亚克隆入TRV介导的基因沉默载体pYL156,以番茄tPDS基因的VIGS沉默体系为对照,对Cf-9和Cf-4基因型植株进行沉默分析,发现正对照植株表现白化症状,说明植株体内tPDS基因已被沉默。片段29沉默后出现严重的发育迟缓和矮化现象,而片段14沉默后没有发现明显症状。这说明核糖体蛋白编码基因ACI29可能参与植株的发育调控,而编码类似钙调磷酸酶的磷酸酯酶编码基因ACI14则可能与发育无关。采用基于cDNA-AFLP片段的基因沉默技术快速大量分离鉴定植物抗病相关基因的策略是可行的。(5)分别对cf-9、Avr9/Cf-9、Cf-4和Avr4/Cf-4基因型番茄幼苗在1d、3d和5d的高温培养后进行降温处理,经Northern杂交分析表明,在不存在互补无毒基因的情况下,Cf-9在各处理中均未见表达,而Cf-4的表达量则随着幼苗培育天数和降温时间的延长而增加;当互补无毒基因存在时,Cf基因的表达强于只含Avr或Cf的对照小苗。两者的表达均随幼苗培育天数和降温时间的的延长而迅速增加。降温处理后,Cf-9进入大量表达的时期早于Cf-4,但Cf-4的表达峰值明显高于前者。这说明发育、温度和无毒基因与Cf基因的表达密切相关,且Cf-9和Cf-4对这些因子影响的反应无论从时序上还是强度上均有区别。更多还原

【Abstract】 The resistance of tomato(Lycopersicon esculentum Mill.)against the leaf mould fungal pathogen Cladosporium fulvum is initiated by the recognition of C.fulvum arivulence(Avr)elicitors by tomato Cf resistance gene products,which activates the downstream signaling,resulting in defense-related gene expression.Over 91%of the amino acids of Cf-9 and Cf-4 proteins are identical,whereas no significant sequence homology is found between Avr9 and Avr4 proteins.However Avr9/Cf-9-and Avr4/Cf-4-induced hypersensitive cell death in tomato seedlings is distinct in the time of initiation,the speed of development and the tissues that HR occurs.The HR in cotyledons of Avr9/Cf-9 seedlings initiates earlier,spreads slower,developing more scattered spots,less associated with veins,when compared with that of Avr4/Cf-4 seedlings.The different HR could be caused by the different expression of the Cf genes and/or downstream signal transduction.To test the hypothesis,this thesis research employed the Avr/Cf tomato seedlings to compare the Avr/Cf-dependent HR developed after the growth temperature shift from 33℃to 20℃,investigate the transcript profiles upon the HR development through cDNA-AFLP, and isolate and clone the TDFs(transcript derived fragments)differentially expressed in the HR~+ and the HR~- seedlings.Expression of some TDFs was assayed in detail.System of TRV-induced gene silencing based on the cDNA-AFLP TDFs to identify the role in resistance of the genes corresponding to the TDFs,was established.In addition,the effect of development,temperature and the cognate Avr on the expression of Cf-9 and Cf-4 was comparatively studied.In order to make deep insights into the molecular mechanism of Cf-9-and Cf-4-dependent HR and resistance,and make a good basis on the future studies on identification and isolation of plant resistance-related genes employing the strategy based on gene silencing using the recombinant silencing vector directly inserted with the cDNA-AFLP TDFs.The main results are as follows:(1)The HR in Avr9/Cf-9 and Avr4/Cf-4 seedlings was compared through microscopic analysis.It was found that high temperature(33℃)attenuates the HR development in the Avr/Cf tomato seedlings. Some cells were stained blue by the dye trypan blue 4 hr and 16 hr post temperature shift from 33℃to 20℃in the Avr9/Cf-9 and Avr4/Cf-4 seedlings,respectively.More and large stained cell patches were observed 12 hr and 48 hr post temperature shift in the Avr/Cf seedlings,respectively.This result indicates that the HR is initiated 4 hr and 16 hr post temperature shift,and typical HR spots develop 12 hr and 48 hr post temperature shift in Avr9/Cf-9 and Avr4/Cf-4 seedlings,respectively.(2)cDNA-AFLP analysis conducted for the seedlings carrying a single Avr gene or Cf gene or a matching Avr/Cf gene pair sampled both at 33℃and after a temperature shift to 20℃.Transcript profiles were compared in HR~+ and HR~- seedlings.TDFs differentially expressed in the HR~+ and the HR~- seedlings were cloned and sequenced.Over 279 480 bands for totally 12 samples appeared in the cDNA-AFLP assay.Among these,367 ACE((?)vr/(?)f-(?)licited,either induced or repressed)TDFs shown differential expression in HR~+ and HR~- seedlings,among which 249 shown induced expression whereas 118 shown reduced expression in both HR~+ Avr9/Cf-9 and Avr4/Cf-4 seedlings.189 TDFs showing most significant differential expression in HR~+ and HR~- seedlings,were cloned and sequenced.The sequences were deposited at GenBank nucleotide sequence database under the accession numbers CD579099-CD579164, CK348286-CK348390,and DN956215-DN956225.The genes corresponding to the 189 TDFs encodes a resistance protein and proteins involved in HR/cell death,signal transduction,defense response,transcription regulation,metabolism and protein synthesis.In addition,9 full-length cDNA sequences were obtained,and named ACI genes for(?)vr/(?)f-(?)nduced genes.These sequences were deposited at GenBank under the accession numbers DQ056433-DQ056442.Among them,ACIs 14 25, 29,36,37 and 39 encodes a calcineurin-like phosphoesterase family protein,a xyloglucan-specific fungal endoglucanase inhibitor protein,a 60S ribosomal protein L10,an oxoglutarate/malate translocator,a cytochrome b559 and a cytoplasmic ribosomal protein S13,respectively,whereas ACIs 13,19 and 112 codes for proteins with unknown function.Results of transcript profiling assay revealed that although all 367 ACE TDFs showed the same trend(either induced or repressed)of differential expression between HR~+ and HR~- seedlings(either Avr4/Cf-4-or Avr9/Cf-9-dependent),42.8%of the ACE TDFs(157/367 in total)showed quantitatively different expression in the two types of HR~+ seedlings.The majority of these(135/157,86.0%)displayed significantly greater differential expression (either induced or repressed)in Avr4/Cf-4 HR~+ seedlings than in Avr9/Cf-9 HR~+ seedlings.These results demonstrate that the spectrum of the genes involved in the Cf-9 and Cf-4 downstream signaling may be identical;however,the fine-tuned regulation of these genes might be different during development of Avr9/Cf-9-and Avr4/Cf-4-dependent HR and resistance.(3)The expression of ACI29 upon development of HR and under stress conditions was analyzed.It was found that in the seedlings carrying a single Avr or Cf gene,and thus showing no HR,AC129 was weakly expressed in the 3d-old and younger seedlings,but strongly expressed in the 4d-old and older seedlings.Expression was stronger in the seedlings carrying a matching Avr/Cf gene pair and thus able to develop HR,and differed in the time-course and profundity in Avr9/Cf-9 and Avr4/Cf-4 seedlings.It was expressed earlier but less strongly upon development of HR in Avr9/Cf-9 seedlings compared with that in Avr4/Cf-4 seedlings,indicating that development and HR affects the expression of ACI29.In addition,expression of ACI29 was weakly induced by heavy metal treatments,strongly repressed by salt and drought treatments,but not affected by wounding treatments.Taken together,these results reveal that the ribosomal protein L10 may be involved in regulation of development,HR and stress response in plants.(4)TDFs 14 and 29 and a tPDS fragment as a positive control were subcloned into TRV-derived gene silencing vector pYL156,respectively.Silencing analysis was conducted in Cf4 and Cf9 tomato plants.Photobleaching phenotype was observed in the plants infiltrated with Agrobacterium transformed with pYL156::tPDS,demonstrating that the endogenous PDS gene has been silenced in these plants.The plants in which the gene corresponding to TDF 29(supposed to be ACI29)was silenced shown severe development retardation and dwarfing,whereas the plants in which the gene corresponding to TDF 14 (supposed to be the ACI14)was silenced,did not shown any visible symptom.This result reveals that this gene silencing system works for cDNA-AFLP TDFs.In addition,ACI29,the ribosomal protein L10, may be involved in regulation of development in plants;while ACI14,a calcineurin-like phosphoesterase family protein,may be not.These results indicate that the strategy employing gene silencing using the recombinant silencing vector directly inserted with the cDNA-AFLP TDFs,to rapidly dentify and isolate the resistance-related genes of plants in large quantities,is feasible.(5)The seedlings,carrying a single Avr gene or Cf gene or a matching Avr/Cf gene pair,were grown at 33℃for 1d,3d and 5d,respectively,and then were grown at 20℃.Results of Northern analysis shown that in the Cf seedlings without carrying a cognate Avr gene expression of the gene Cf-9 was not detected,whereas expression of the gene Cf-4 was obvious.The expression of Cf-4 gradually enhanced with the increasing growing days maintained at 20℃and the longer growing hours after the temperature shift.In Avr/Cf seedlings,however,both Cf genes were expressed more strongly than in Cf seedlings.The expression of both Cf genes rapidly strengthened with the increasing growing days maintained at 20℃and the longer growing hours after the temperature shift.After temperature shift,the expression of Cf-9 was more rapidly but the peak level was lower compared with that of Cf-4.These results demonstrate that development,temperature and the cognate Aw affect the expression of both Cf genes,but to a different extent.更多还原