【作者】 李亚军；

【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】 2008年被联合国宣布为“国际马铃薯年”,彰显出马铃薯作为世界上第四大粮食作物在粮食安全和消除贫困中所具有的至关重要的作用。由致病疫霉[Phytophthorainfestans(Mont.)de Sary]引起的晚疫病作为第一大粮食作物病害,在世界各地流行频繁,已成为制约全球马铃薯生产和发展的重要因素。化学药剂防治晚疫病的手段由于引起环境污染、农药残留以及抗药生理小种的产生等一系列问题而受到诟病,利用马铃薯自身的防御机制来提高抗病能力已成为晚疫病防治的一种新思路。主效R基因控制的垂直抗性很容易被新的病原菌生理小种克服,由微效多基因控制的水平抗性因其抗性稳定持久,越来越受到育种家的亲睐。马铃薯对晚疫病的抗性还能经外界生物或非生物因子所诱导。β-氨基丁酸(β-aminobutyric acid,BABA)即能诱导这种抗性,且诱导的抗性具有水平抗性的特征。然而目前对水平抗性和β-氨基丁酸诱导抗性的机理研究还很薄弱,极大限制了抗性机制的深入研究和抗性资源的充分利用。本研究旨在通过比较水平抗性和BABA诱导抗性差异表达基因的异同,了解马铃薯晚疫病水平抗性与诱导抗性之间的关系,探索BABA诱导抗性和晚疫病水平抗性产生的分子机理。取得的主要研究结果如下:1.以剔除了来源于Solanum demissum的R1-R11的马铃薯植株为材料,喷施不同浓度的BABA,然后接种P.infestans,结果表明2mmol/L和4mmol/L浓度的BABA预处理3d和4d能有效诱导马铃薯对晚疫病的抗性。2.利用cDNA-AFLP技术筛选到101个在马铃薯水平抗性和BABA诱导抗性(BABA-IR)产生过程中差异表达的片段,其中49个差异片段为P.infestans和BABA共同诱导表达,说明BABA在很大程度上通过激活植物防卫体系来表达诱导抗性。并且多数共同诱导表达的片段对BABA的响应速度更快,说明诱导植物防卫基因的快速表达是BABA-IR的重要机制。NCBI数据库和TIGR马铃薯TC数据库检索结果表明,共同诱导表达的差异片段的同源基因参与抗病反应的各个方面,包括与信号传导和转录调控有关的基因以及与植保素和生物碱生物合成相关的基因等。利用病毒诱导的基因沉默体系初步验证两个差异片段在诱导抗性中起重要作用,进一步证明所筛选的差异表达基因确实参与了植株对晚疫病抗性的反应。3.部分筛选出的差异表达片段与参与SA和JA信号传导途径相关的基因同源,表明SA和JA依赖的信号传导途径可能参与到BABA诱导抗性产生的过程中来。这些信号传导相关的差异片段同样能被P.infestans诱导表达,意味着BABA启动了植物本身所具有的抵御病原入侵的信号途径,马铃薯BABA诱导抗性与水平抗性的抗性机制存在更为广泛的交联。4.利用电子克隆和RACE相结合的方法克隆了一个晚疫病菌和BABA共同诱导表达的基因StWRKY5的全长编码序列,推测编码297个氨基酸,3′端包含339bp的非编码序列。预测蛋白的结构显示StWRKY5含有WRKYGQK的保守基序和C2H2型锌指基序,该基因可能为马铃薯WRKY转录因子家族中的一个成员,GenBank登记号为EU056917。Southern杂交结果显示StWRKY5在马铃薯基因组中存在1-2个拷贝。半定量RT-PCR结果表明,P.infestans、BABA和水杨酸在处理后4h即可诱导该基因的上调表达;茉莉酸甲酯和伤害分别在处理后12h和24h才开始诱导该基因的表达。这些工作为进一步深入研究StWRKY5的功能奠定了基础。更多还原

【Abstract】 The year 2008 has been declared the International Year of the Potato by the United Nations,noting that potato,as the fourth largest crop species,can play vital roles in facing food security and eradicating poverty.Potato late blight,caused by Phytophthora infestans(Mont.) de Bary,ranks the most destructive disease in world agriculture,has seriously limited the potato production and development.Current disease control in the field depends on repeated application of large amounts of fungicides.However,this strategy resulted in environmental problems,pesticide residue and evolution of new races of P.infestans with new virulence alleles.Triggering the host plant’s defense system to defend itself against pathogen attack seems to be quite prospective.Since the major gene resistance is easy to be overcome by new isolates of P.infestans in potato,horizontal resistance become "a great value" due to its durable and stable performance.In addition, plants also employ inducible resistance mechanisms which can be triggered by a variety of biotic and abiotic stimuli.A non-protein amino acid DL-β-amino-butyric acid(BABA) is capable of protecting numerous plant species against various pathogens.Moreover, BABA-induced resistance(BABA-IR) possesses a feature like the horizontal resistamce which is also durable and stable.However,to date,we know little about the molecular processes of horizontal resistance and BABA-IR,which greatly limits the in-depth study of the resistance mechanism and full use of the resistant resources.In this study,we compares gene expression profiles of a potato clone harboring horizontal resistance to P. infestans in response to BABA and the pathogen by using cDNA-AFLP,aiming at foundational elucidation of molecular mechanisms of BABA-IR and horizontal resistance. The main results are as follows:1."R-gene-free" potato plants were sprayed with BABA at concentration of 0.5mmol/L, 1mmo/L,2mmol/L and 4mmo/L.After 1d、2d、3d and 4d,the leaves were detached and inoculated with P.infestans.The results indicated that potato plants pre-treated with 2mmol/L or 4mmo/L BABA exhibited a significantly higher level resistance to P. infestans compared to the control.2.In total,101 transcript derived fragments(TDFs) showed significant differential expression among the BABA treated samples,P.infestans inoculation and their respective control ones.About half of differentially expressed fragments(49) were overlapped in both BABA and P.infestans induction systems indicating BABA,to a large exrend,induces plant resistence through activating the plant denfence system.Most of the TDFs in common expressed earlier in response to BABA than in response to the pathogen implying early activation of the plant defence system is a main mechanism of BABA-IR. Homology analysis showed that these TDFs involved in signalling,cell wall strengthening and synthesis of antimicrobial compounds.Two TDFs were verified to play important roles in tobacco induced resistance using vires-induced gene silencing,fuether approving the genes selected are involved in response to the pathogen attack.3.Several differentially expressed TDFs were similarities to genes involved in SA and JA dependent signal pathways,indicating that the two signal pathways play roles in BABA-IR.These TDFs related to signaling were also up-regulated by P.infestans, demonstrating that the signal pathway controlling pathogen induced could be triggered by BABA,and implying the extent crosstalk between BABA-IR and horizontal resistance.4.The CDS sequence of gene StWRKY5 was cloned using the method of combination with Silico cloning and 3’-RACE.GenBank accession number is EU056917.The gene contains 339 bp 3’-UTR region and the peptide sequence was predicted to be 297 amino acids.The deduced StWRKY5 protein would be WRKY transcription factor due to it contains conserved WRKYGQK motif and C2H2 zinc finger motif.Southern blot showed potato genomes possess 1-2 copies of StWRKY5.Semi-quantitative RT-PCR analysis showed that StWRKY5 were induced at 4 h by P.infestans,BABA and SA,while it expressed at 12 h and 24 h after MJA and wound,respectively.These efforts made the foundation to further study the function of StWRKY5.更多还原