【作者】 刘辉；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2012， 博士

【摘要】 番茄CSolanum lycopersicum)属非冷驯化植物,在生长发育的各个阶段都容易遭受低温伤害。提高番茄的抗寒性可降低低温对植株及果实的伤害,延长生长周期,减少设施栽培的投入等。筛选番茄耐低温材料,培育抗寒番茄新品种具有重要的实际应用价值。CBF低温应答途径是目前研究最为清楚,也是冷驯化植物最重要的低温应答调控途径。番茄中同样存在CBF低温响应途径,但其在低温应答中发挥的作用较小。非冷驯化的番茄对低温适应的分子机制可能不同于冷驯化植物。野生多毛番茄(S. habrochaites)的抗寒性显著高于栽培番茄。过去几十年里,对多毛番茄和普通番茄在低温胁迫下的生理生化差异进行了大量研究,并取得了重要进展。但关于多毛番茄和普通番茄在低温胁迫下的基因表达差异,多毛番茄抗寒的分子机制知之甚少。本研究以多毛番茄LA1777、栽培番茄LA4024及由二者渐渗系(IL)群体为材料,对苗期植株抗寒性进行评价和筛选。为揭示番茄抗冷的分子机理,利用TOM2基因芯片对抗寒材料(供体亲本LA1777和渐渗系LA3969)和冷敏感材料(受体亲本LA4024)在低温胁迫下的基因表达差异进行了分析。根据芯片分析结果,从多毛番茄中克隆了3个抗寒相关基因,并进行了功能鉴定。主要研究结果如下：1.鉴定了多毛番茄中贡献抗寒性的染色体区段：通过比较多毛番茄LA1777和普通番茄LA4024在低温胁迫表型及相关生理指标差异,建立了番茄抗寒性鉴定体系。利用该体系对LA1777渐渗系群体幼苗进行耐低温鉴定,发现22个IL系在低温(4℃)处理3d时萎焉程度较受体亲本LA4024轻,在多毛番茄的第1、2、3、4、5、6、7、9、11及12条染色体上可能含有抗寒相关的QTLs。其中,渐渗系LA3969在整个低温处理及恢复过程中具有与供体亲本LA1777相近的表型,抗寒性显著高于受体亲本LA4024及其它IL系。低温胁迫下,LA3969和LA1777细胞膜受伤害程度较轻。低温处理10d恢复一周后,LA3969和LA1777的存活率显著高于LA4024。LA3969的抗寒性来源于导入了野生多毛番茄的第12条染色体片段。这表明在多毛番茄第12条染色体上可能含有一个或多个控制抗寒性的关键QTL/基因。2.抗/感番茄材料低温应答的分子差异：利用TOM2芯片分析了LA1777、 LA3969和LA4024幼苗在低温胁迫下的基因表达差异。低温胁迫处理3d后,在LA1777、LA3969和LA4024中分别鉴定了1613、1456和1523个低温应答基因。其中,103个低温应答基因仅在LA1777和LA3969检测到,196个低温应答基因仅在LA4024中检测到。这些基因可能在决定番茄对低温的抗或敏感性中起着重要作用。功能聚类分析表明,更多与逆境应答、内源刺激应答、信号转导、转录调控、生物合成、次生代谢、蛋白代谢等相关的基因在两抗性材料中上调表达。而更多与光合作用相关的基因在冷敏感材料中被抑制。GO (Gene Ontology)生物学进程富集分析发现更多的生物学进程在两抗性材料中被加强,而更多的生物学进程在冷敏感材料中被抑制。低温胁迫下,茉莉酸生物合成、油菜素内酯代谢过程、苯丙素生物合成、淀粉降解、亮氨酸生物合成、卡尔文循环和超氧自由基清除等7个代谢途径在抗感材料间发生了显著调整。3.番茄抗寒的分子机制：差异统计分析表明,92个基因在两抗性材料与冷敏感材料间表达变化差异显著。126个基因在LA1777中的表达变化显著不同于LA3969和LA4024。这些基因基因可能在贡献LA3969和/或LA1777的抗性中起着重要作用。对这218个差异表达基因进行染色体定位分析,发现80个基因被定位到22个筛选的抗性渐渗系染色体替换区段或已报道的多毛番茄抗寒相关QTLs区域。其中,11个位于LA3969染色体渗入的片段上,这些基因可能在贡献LA3969的抗寒性中起着重要作用。GO生物学进程富集分析表明差异表达基因中许多参与了钙信号调控、激素和ROS的动态平衡调节及信号应答。钙离子、激素及ROS作为信号分子可能在调控番茄低温应答中具有重要作用。这些信号途径在抗感材料间的调整引起下游一系基因表达的改变,包括转录因子(如HSFs、MYBs、NACs等)、翻译后修饰蛋白(如SKP2A、LAP-A1、XERICOs等)、功能蛋白(如HSPs、PRs及脱水素等)、代谢相关的酶(如GSTs、LOXs、BAM等)等。这一系列基因表达的改变使得LA1777和LA3969的抗寒性显著较LA4024提高。4.差异表达基因ShDHN的功能鉴定：根据芯片分析结果,从多毛番茄中克隆了一脱水素基因,命名为ShDHNo芯片及实时定量PCR分析表明,该基因的表达受低温胁迫诱导,且在抗性材料中的表达高于冷敏感材料。同时,该基因也受干旱、高盐、渗透胁迫、ABA和JA诱导。在普通番茄LA4024中超量表达ShDHN显著提高了植株的抗寒和抗旱性,促进了幼苗在高盐及渗透胁迫下的生长发育。同野生型相比,转基因株系在低温和干旱胁迫下积累更多的脯氨酸,具有更高的SOD和CAT活性,逆境条件下质膜的伤害程度降低。低温胁迫下,转基因株系植株叶片中H2O2和O2-的积累明显少于野生型植株。超量表达ShDHN提高了SOD1、GST和PRl的表达,降低了POD、LOX和PR2的表达。这些结果表明ShDHN可能通过提高植株在逆境条件下ROS的清除能力、促进渗透调节物质的积累、调节其它信号途径及相关基因的表达来增强植株对非生物逆境的抗性。5.差异表达基因ShCHL P的功能鉴定：根据芯片分析结果,从LA1777中克隆了一牻牛儿基牻牛儿基还原酶基因,命名为ShCHL P。组织表达谱分析表明,该基因在叶和茎中表达量较高,在根中基本不表达。干旱、高盐、低温、高温及氧化胁迫下,该基因的表达被显著抑制。我们构建了ShCHL P超量表达的载体,并转化普通番茄LA4024。在获得超量表达株系的同时,也发现了几个共抑制株系。超量表达ShCHLP提高了植株叶片叶绿素含量,促进了幼苗在正常、高盐及渗透胁迫下的生长发育；而共抑制株系植株叶片黄化,茎、叶及果实中的叶绿素含量明显降低,幼苗在高盐及渗透胁迫下的生长发育显著受到抑制。超量或抑制该基因的表达均降低了氧化胁迫对植株的伤害,导致这种表型的分子机制有待于进一步分析。这些结果表明,CHLP是植物叶绿素生物合成所必需的,该基因在植物生长发育及非生物逆境应答中起着重要作用。6.差异表达转录因子ShNAC的功能鉴定：根据芯片结果,从LA1777中克隆了一NAC转录因子,命名为ShNAC。该基因编码405个氨基酸,在其蛋白N端有一NAM保守结构域。ShNAC在LA1777各组织中呈组成型表达,以果实和花中的表达量最高,在茎中的表达最低。低温、干旱和高盐胁迫均能诱导该基因的表达。正常生长条件下,超量表达ShNAC转基因植株较野生型LA4024植株变矮,且茎基部变软,植株不能直立。超量表达ShNAC转基因植株较野生型对低温和干旱更敏感。ShNAC可能是植物生长发育及非生物逆境应答的一个负调节因子。更多还原

【Abstract】 The cultivated tomato(Solanum lycopersicum) is unable to cold acclimate, and easily suffers cold injury at all stages of plant growth and development. Several practical benefits of increased cold tolerance in cultivated tomato would be:prevention of plant and fruit damage from cold stress, extension of the growing season, and reduction of input of facility cultivation. Thus, screening of cold tolerant tomato resources, and breeding new varieties that can tolerate cold stress have important practical value.The C-repeat binding factor (CBF) cold response pathway is currently the best documented system that plays a pivotal role in gene regulation during cold acclimation. Tomato also has the CBF cold response pathway, but its function appears to be considerably smaller. The molecular basis of cold adaptation in non-cold-acclimated tomato may differ from that of the cold-acclimated plants.The wild tomato S. habrochaites is more tolerant to low temperature than cultivated tomato. Significant progress has been made in the past decade in elucidating the differences in physiological responses under cold stress between S. habrochaites and S. lycopersicum. However, the differences in global gene expression under cold stress between the two genotypes and the molecular mechanisms responsible for cold tolerance in S. habrochaites are largely unknown.In this study, seedlings of S. habrochaites LA1777, S. lycopersicum LA4024, and introgression lines (ILs) of them, were evaluated for their tolerance to low temperature. To explore the molecular mechanisms of cold tolerance in tomato, the TOM2array was used to compare the transcriptome differences between the tolerant genotypes (the donor parent LA1777and the sleeted IL LA3969) and the sensitive one (the recurrent parent LA4024) under cold stress. Based on the microarray results, three cold-responsive genes were cloned from S. habrochaites and functionally analyzed. The main results are as follows:1. Identification of chromosomal regions conferring cold tolerance in S. habrochaites. A system for cold tolerance evaluation of the tomato seedlings was developed by comparing phenotypic and physiological responses of seedlings of S. habrochaites LAI777and S. lycopersicum LA4024under cold stress. Seedlings of LAI777ILs were evaluated under low temperature (4℃) using this system. Twenty-two ILs with S. habrochaites introgressions on chromosomes1,2,3,4,5,6,7,9,11, and12exhibited less severe wilting than LA4024after3d of cold treatment. Among these, the phenotypic performance of LA3969was quite close to that of LA1777during cold stress and recovery, and showed stronger cold tolerance than LA4024and other ILs. LA3969and LA1777suffered less membrane damage during cold stress and showed significantly higher survival rates than LA4024after10d of cold stress and recovery for one week. LA3969contains a large introgressed segment from S. habrochaites on chromosome12. This indicates that at least one major QTL/gene responsible for cold tolerance is located on S. habrochaites chromosome12.2. The molecular differences between tolerant and sensitive tomato in reponse to cold stress. Transcriptome analysis of LA1777, LA3969, and LA4024seedlings under cold stress were performed using TOM2array. After3d of cold stress (4℃), a total of1613,1456, and1523cold-responsive genes were identified in LA1777, LA3969, and LA4024, respectively. Among these,103cold-responsive genes were exclusively identified in both LA1777and LA3969, whereas196cold-responsive genes were uniquely observed in LA4024. These genes may play important roles in conferring tolerance or sensitivity to chilling in tomato. Functional classification of cold responsive genes showed that more genes involved in ’response to stress’,’response to endogenous stimulus’,’signal transduction’,’transcription’,’biosynthetic process’,’secondary metabolic process’, and ’protein metabolic process’ were up-regulated in the two tolerant genotypes, whereas more genes involved in photosynthesis were down-regulated in the sensitive genotype. Gene ontology (GO) term enrichment analysis revealed that more GO biological process terms were significantly enriched among the up-regulated genes in the two tolerant genotypes, whereas more biological processes were significantly repressed by cold stress in the sensitive one. A total of7biochemical pathways varied significantly between tolerant and sensitive genotypes under cold stress, including jasmonic acid biosynthesis, brassinosteroid metabolic process, phenylpropanoid biosynthesis, starch degradation, leucine biosynthesis, Calvin cycle, and removal of superoxide radicals.3. The molecular mechanisms of cold tolerance in tomato. A total of92cold-responsive genes with statistically significant differences in expression between the two tolerant and sensitive genotypes were identified. In addition, the expression of126 genes in L1777showed significantly different from that in LA3969and LA4024under cold stress. These genes may play important roles in conferring cold tolerance in LA3969and/or LA1777. Among these,80genes were located on the introgressed chromosomal segments of the22selected cold-tolerant ILs and/or cold tolerance QTLs identified previously in S. habrochaites. Of these,11genes were localized to the introgressed chromosomal segment of LA3969. These genes may play critical roles in conferring cold tolerance in LA3969. GO term enrichment analysis showed that many genes involved in calcium-mediated signaling, ROS and hormone homeostasis were differentially expressed. Calcium, ROS, and hormones as signaling molecules may play important roles in regulating gene expression in response to cold stress in tomato. The modulation of these signaling pathways caused differential expression of many transcripts between tolerant and sensitive genotypes under cold stress, including transcription factors (e.g., MYBs, HSFs, and NACs), post-translational modification proteins (e.g., SKP2A, LAP-A1, and XERICOs), functional proteins (e.g., HSPs, PRs, and dehydrin), and metabolic enzymes (e.g., GSTs, LOXs, and BAM), and etc. These specific modifications make LA1777and LA3969more cold tolerant than LA4024.4. Functional characterization of differentially expressed gene ShDNN. Based on the microarray results, a dehydrin gene was isolated form S. habrochaites and designated ShDHN. Both microarray and quantitative real-time RT-PCR analysis indicated that the expression of this gene was more strongly induced by cold stress in the tolerant genotype than the sensitive one. In addition, ShDHN expression was also induced by drought, salt, osmotic stress, ABA, and MeJA. Overexpression of ShDHN in LA4024increased tolerance to cold and drought stress, and improved the early seedling development under salt and osmotic stress. Compared with the wild type, the transgenic lines accumulated more proline, maintained higher activities of SOD and CAT, and suffered less membrane damage under cold and drought stress. Under cold stress, the accumulation of H2O2and O2-was less in the transgenic plants than in the wild type. The expression of SOD1, GST, and PR1were increased in the ShDHN overexpression lines, while the transcripts of POD, LOX, and PR2were inhibited. These results indicate ShDHN confers abiotic stress tolerance by enhancing ROS scavenging capacity, accumulating higher amounts of compatible solutes, and regulating other signaling pathways and genes expression.5. Functional characterization of differentially expressed gene ShCHL P. Based on the microarray results, a geranylgeranyl reductase gene, was isolated from LA1777and designated ShCHL P. ShCHL P is highly expressed in the leaf and stem, and nearly no expression in root. Its expression was suppressed by drought, salt, low or high temperature, and oxidative stress. The overexpression vector of ShCHL P was constructed and translated into the cultivated tomato LA4024. Interestingly, we got both overexpression and co-suppression of CHL P in transgenic tomato plants. Overexpression of ShCHL P increased the leaf chlorophyll content, improved the early seedling development under normal, slat and osmotic stress conditions. Whereas the leaves of the co-suppression lines were yellow, the contents of chlorophyll in leaf, stem, and even fruit were decreased, and the early seedling development was significantly inhibited in co-suppression lines under slat and osmotic stress conditions. Both overexpression and co-suppression of CHL P transgenic plants enhanced oxidative stress tolerance. The molecular machines lead to this need to be further analysis. The results indicate that CHL P is required for chlorophyll biosynthesis, and it plays an important role in growth and development, and abiotic stress response in plants.6. Functional analysis of differentially expressed transcription factor ShNAC. Based on the microarray results, a NAC transcription factor (ShNAC) was isolated from LA1777. ShNAC encodes a protein of405amino acids, which has a conserved NAM domain in N-terminal. ShNAC is constitutively expressed in various tissues of LA1777, and the expression in fruit and flower is the highest, whereas the transcript abundance in stem is the lowest. Under normal growth condition, the plant height of ShNAC overexpression lines was significant lower than that of the wild type. The bottom of stem of the transgenic plant was flexible, and the plant was unable to stand erect. The ShNAC transgenic plants were hypersensitive to drought and cold stress compared with wild type. The results indicate ShNAC may act a negative regulator of growth and development, and abiotic stress responses in tomato.更多还原