【作者】 倪雪梅；

【导师】 谢从华；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2009， 博士

【摘要】 泛素-蛋白酶体通路在植物防卫反应(包括过敏性反应)中发挥重要作用,泛素连接酶(ubiquitin ligase, E3)则是参与这一过程的重要组分。本研究旨在通过克隆和分析与马铃薯晚疫病抗性相关的泛素连接酶家族中的成员,初步揭示这类基因在马铃薯晚疫病垂直抗性和水平抗性中的调控作用,或它们在马铃薯防御晚疫病菌侵染的信号传导网络中的作用,为进一步深入开展抗性机理研究奠定基础。本实验室从晚疫病诱导的马铃薯水平抗性材料构建的cDNA文库中筛选出两个与烟草Avr9/Cf-9 rapidly elicited protein (ACRE,具有泛素连接酶E3活性)基因有很高同源性的EST片段(10-A12和08-E12)。本研究中,我们克隆了这两个EST片段所代表的基因,StRFP和StPUB,并研究了其生物学功能。StRFP基因的全长cDNA为1354 bp,包含一个789 bp的开放阅读框(ORF),编码262个氨基酸,含有高度保守的RING-H2型RING指结构域、跨膜结构域和GLD结构域,是一个新的马铃薯ATL (Arabidopsis toxico para levadura)蛋白。StRFP基因位于马铃薯第3条染色体上,不含内含子。马铃薯基因组中可能有1-2个StRFP基因拷贝或与之同源性高的基因。利用洋葱表皮细胞进行的StRFP-GFP融合蛋白亚细胞定位结果显示,StRFP在细胞膜上或膜外表达。RT-PCR分析结果表明,StRFP基因表达无组织特异性,在根、茎、叶、花、茎尖、匍匐茎和块茎等各种组织器官中组成型表达,但不同部位的表达水平有差异。离体叶片的晚疫病原菌接种表明,StRFP在接种24 h后表达增强,水杨酸(SA)、茉莉酸甲酯(MeJA)、脱落酸(ABA)和机械伤害处理能诱导其表达,乙烯(ETH)不能诱导其表达。试管苗在外界环境胁迫下的表达模式表明,StRFP对所研究的各种环境因素如NaCl、20%PEG、脱水、40℃热激和4℃低温都有响应,但对盐胁迫和渗透胁迫的反应更明显。为了进一步研究其功能,我们分别构建了CaMV 35S驱动下的StRFP基因正义表达载体和RNAi抑制表达载体,采用根癌农杆菌介导法转化鄂马铃薯3号(EP3)的试管薯,分别获得超量表达株系27个和干涉株系6个。Southern杂交表明每个转基因植株有1-3个拷贝。晚疫病病原菌接种实验表明,与未转基因对照相比,超量表达的转基因株系更抗病,而抑制表达的株系更感病,说明StRFP基因为马铃薯抗晚疫病反应中的正向调控因子。StPUB基因全长2483 bp,包含一个2175 bp的ORF,编码724个氨基酸,GenBank登记号为EF091878。序列分析表明,StPUB蛋白具有保守的UND/U-box/ARM结构域,与烟草ACRE276的氨基酸序列有89%的同源性。从马铃薯水平抗性材料(386209.10)、垂直抗性材料(鄂马铃薯3号,EP3)和感病材料(转心乌,ZXW)中获得StPUB基因的基因组DNA信息,序列分析表明该基因不含内含子,且在三种不同抗性材料中大小一致,同源性达到97.7%。通过TAIL-PCR与生物信息学分析,推测StPUB基因包含三个启动子序列,可能位于马铃薯第2条染色体上。Southern blot杂交结果表明,马铃薯基因组中可能有2-3个StPUB拷贝或与之同源性高的基因。离体叶片的诱导表达模式研究表明,晚疫病病原菌接种24 h后,StPUB表达明显增强,而且在三种不同的马铃薯抗性材料中表达趋势一致；SA、ABA和机械伤害能激活StPUB表达,而MeJA和ETH不能诱导其表达。试管苗在外界环境胁迫下的RT-PCR结果显示,热激(40℃)、低温(4℃)、20%PEG或NaCl能诱导StPUB表达。采用RNAi技术,构建了抑制StPUB基因表达的载体,并利用农杆菌介导的遗传转化法导入马铃薯垂直抗性品种EP3中,共获得卡那霉素抗性转化植株13株。PCR检测和Southern杂交结果表明,外源基因已经整合到马铃薯基因组中,其拷贝数1-3个不等。对转基因植株中StPUB基因的表达进行RT-PCR分析表明,StPUB基因在转基因植株中的表达量比对照显著降低,RNAi能有效的抑制StPUB基因的表达。从晚疫病病原菌离体接种结果发现,沉默StPUB的转基因株系,其病斑生长速率(LGR)比非转基因对照高,表明沉默StPUB基因降低了马铃薯的抗病性。转基因株系试管苗耐盐性试验结果表明,在盐胁迫条件下,沉默StPUB的转基因植株与野生型植株相比,地上部分和根系生长相对较弱,根部腐烂的株数明显增多,表明抑制.StPUB表达降低了马铃薯转基因株系的耐盐性。上述结果表明,StPUB在激活马铃薯抗晚疫病及耐盐反应中起作用。更多还原

【Abstract】 The ubiquitin-proteasome pathway plays a central role in plant defense responses (including hypersensitive response), the specificity of whose is controlled by E3 ubiquitin ligases which selectively recognize the appropriate protein substrate for breakdown. The aim of the studies reported in this paper was to clone and analyze the ubiquitin ligases-related genes to look into the regulating function of them associated with vertical and horizontal resistance to late blight, or the signal transmitting function of potato prevent late blight infection, and provision of fundamental bases for further study.Two expressed sequence tags (ESTs) (10-A12 and 08-E12) which are similar to tobacco (Nicotiana tabacum) Avr9/Cf-9 rapidly elicited (ACRE) genes were firstly identified from Phytophthora infestans-inoculated leaves of potato (Solanum tuberosum L.) clone 386209.10 which is free from R1-R11 genes. In present study, we cloned two genes standing for these two ESTs, StRFP and StPUB, and elucidated possible functions of them responding to biotic and abiotic stresses.The full-length cDNA of StRFP is of 1354 bp with an open reading frame (ORF) of 789 bp which encodes 262 amino acid residues. The deduced amino acid sequence contains a highly conserved RING-H2 type RING finger domain, a transmembrane region and a GLD signature, implying StPUB is a novel ATL (Arabidopsis toxico para levadura) protein of potato. The Southern blot analysis and mapping indicated that StRFP had one to two copies in potato genome without intron locating on chromosome 3. Transient expression of StRFP:GFP in onion epidermal cells revealed that StPUB localized to the plasma membrane or out of that. RT-PCR assays showed that StRFP was constitutively and differentially expressed in potato plants, including roots, stems, leaves, flowers, shoot tips, stolons and tubers, and significantly induced in potato detached leaves treated with P. infestans, a pathogen causing potato late blight, and plant defense-related signal molecules such as salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA), implying StRFP may be involved in potato response to pathogen attack. The results of treating in vitro potato plantlets with environmental stresses showed that StPUB was responsive to NaCl,20% PEG, dehydration,40℃heat and 4℃cold, especially enhanced salt and osmosis tolerance. The function of StRFP in potato resistance against late blight was further investigated by constructing of overexpression and RNA inteference (RNAi) vectors and introducing into potato cv. E-potato 3. There were 27 overexpression and 6 RNAi silencing transgenic lines with 1-3 copies of the gene insertion confirmed by the Southern blot analysis. By challenging the detached leaves with mixture races of P. infestans containing races 1,3,4, and 1.3, all of StRFP-overexpressing transgenic plants displayed a slower disease development than nontansformed control in terms of the lesion growth rate (LGR) and four out of eleven transformants were significant (p<0.05). In contrast, StRFP-silencing lines by RNAi were more susceptible to the pathogen infection and three of four lines showed significantly higher LGR than control. Present results demonstrate that StRFP plays a positive role in the resistance of potato against P. infestans.The full-length of StPUB is of 2483 bp with an ORF of 2175 bp encoding 724 amino acids (GenBank acc. No. EF091878). The StPUB protein contains UND/PUB/ARM repeat domain with 89% of identity to tobacco ACRE276. Furthermore, the intron-exon junction of StPUB gene established by comparison between the cDNA and the genomic sequences from potato cvs.386209.10, E-potato 3 (EP3) and Zhuanxinwu (ZXW) indicated that the sequences maintained high levels (97.7%) of conservation in terms of sequence length and nucleotide composition, and all the gDNAs from the three cultivars seemed to be composed of a single exon without intron. The TAIL-PCR and bioinformatics analysis speculated that StPUB had three promoter sequences and maybe locate on chromosome 2. The DNA gel blot analysis indicated that there are two to three copies of StPUB or closely related genes in the potato genome. The RT-PCR analysis showed a similar expression pattern of StPUB obseverd in the three potato cultivars when detached leaves were infected by P. infestans. The expression of StPUB was upregulated not only in detached leaves by signal molecules of salicylic acid and abscisic acid and wounding, but also in in vitro plantlets by high (40℃) and low (4℃) temperatures and dehydration induced by polyethylene glycol and NaCl. The function of StPUB was further clarified by silencing the gene in cv. E-potato 3 using RNAi-based post-transcriptional gene silencing (PTGS). Thirteen transgenic lines with 1-3 copies of the gene insertion were obtained, and much lower expression of StPUB was observed in transgenic lines than control monitored by RT-PCR. The LGR of StPUB-RN Ai transgenic plants were higher than the control, revealing that silencing of StPUB in potato resulted in a partial loss of plant resistance against late blight. Its function in the salt tolerance was further investigated by subjecting the in vitro plantlets to different concentrations of NaCl stress, transgenic plantlets showed much higher sensitivity to NaCl than control in terms of shoot length, plant fresh weight, rooting ratio and percentage of decayed plantlets, suggesting that StPUB is also involved in response to the salt stress. Present data indicated that StPUB is a gene harboring broad-spectrum responses to both biotic and abiotic stresses in the potato and may play crucial roles in late blight resistance and salt tolerance.更多还原