【作者】 王华忠；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 小麦是我国乃至世界的重要粮食作物。小麦真菌病害是影响小麦产量的重要因素。随着植物分子生物学和功能基因组学的快速发展,我们对植物复杂的抗病反应过程有了越来越清晰的了解。同时,越来越多的抗病和抗病反应过程相关基因的克隆也成为可能。对这些基因进行功能鉴定和抗病效果分析,并利用基因工程手段开展小麦抗病分子育种将具有广阔的前景。结合传统的育种方法,对小麦抗病性状的改良将更为快速和高效。本实验首先利用基因枪转化方法,将两个抗病相关基因TaTBL和TaTST转入到感病小麦品种杨麦158中,获得经过分子鉴定和抗性分析的转化植株。随后,又利用瞬间表达技术对TaTBL、TaPK1、TaTST、G1b3s2、Cht4和G1b3s6等6个抗病相关基因的功能进行了分析,表明其在感病小麦叶片表皮细胞中的过量表达能显著抑制白粉菌对细胞的侵入和吸器的形成,提高表达细胞的抗性。 1.1 小麦抗病相关基因的转化 利用花青素合成酶调节基因作为报告基因对影响基因枪转化的多个参数进行了深入的分析,优化了转化过程。随后,分别构建了两个抗病相关基因小麦类Beclin1基因TaTBL和硫代硫酸硫转移酶基因TaTST的植物高效表达载体,目标基因的启动子为来源于玉米泛素基因的单子叶植物高效启动子ubi,植物选择标记基因为除草剂抗性基因bar。使用基因枪将表达载体分别导入到再生频率较高的感白粉病小麦品种杨麦158的幼胚愈伤组织细胞中。轰击后经过两轮除草剂bialaphos筛选和抗性愈伤组织的再生,分别获得转TaTBL基因的抗性植株50株;转TaTST基因的抗性植株30株。提取抗性植株基因组DNA,分别依据bar基因、Ubi启动子和目标基因的序列设计并合成引物进行PCR扩增,结果分别有6株和5株能够同时正确扩增出bar基因、Ubi启动子和相应的目标基因TaTBL或TaTST基因片断,初步表明外源基因确已整合到了小麦基因组上。转化植株苗期离体叶片的白粉病接种实验表明,TaTBL和TaTST基因的导入,在一定程度上增强了转化植株对白粉菌的抗性,表现为延缓了白粉菌的发育。 1.2 小麦抗病相关基因的瞬间表达研究 采用瞬间表达技术分析了小麦类Beclin1基因TaTBL、丝氨酸/苏氨酸激酶基因TaPK1、硫代硫酸硫转移酶基因TaTST、β-1,3-葡聚糖苷酶基因G1b3s2、几丁质酶基更多还原

【Abstract】 Wheat is a member of the Triticae group of cereals and indisputably one of the major food crops of the world and a foundation of human nutrition worldwide. Fungus disease is an important biotic stress that greatly impacts the productivity of wheat. With the fast progress of plant molecular biology and plant functional genomics, the complicated resistance reaction process of plant have been understood much clearly, and more and more resistance genes and related genes have been cloned. To effectively analyze the potential resistance functions of these genes and tranform them into wheat to get resistant cultivars are in great need for crop resistance breeding (molecular breeding). In this experiment, two resistance-related genes TaTBL and TaTST were respectively transformed into wheat susceptible cultuvar Yangmai158 and transgenic plants were identified by PCR analysis. Increased resistance of transgenic plants to powdery mildew was observed after inoculation of wheat powdery mildew pathogen fungus Erysiphe graminis f.sp. tritici conidiospores on detached leaf segments. Besides stable transformation, transient expression system were also performed to identify resistance functions of six resistance-related genes TaTBL TaPK1 TaTSTs Glb3s2, Cht4 and Glb3s6. Over-expression of five of them in wheat epidermal cells can partly inhibit penetration of conidiospores and formation of haustoria, and to some extent increase the resistance of expression cells to powdery mildew.1.1 Transformation of two resistance-related genes into wheat and resistance analysis of transgenic plants.Anthocyanin synthesis regulatory genes Cl/Lc were used to optimize the parameters of gene-gun transformation protocol through counting of red spots on wheat calli after transient expression. Two plant expression vectors respectively carrying the wheat resistance-related genes TaTBL and TaTST driven by the ubi promoter and the bar gene with resistance to PPT were constructed. And then immature embryo derived calli of wheat cultivar of Yangl58 which has a high regeneration efficiency in tissue culture were used to perform particle bombardment. Through two sets of bialaphos screening and regeneration of resistance calli, 50 and 30 bialaphos-tolerrent plants of the two genesrespectively were got out of about 500 initial calli. Genomic DNA were extracted and PCR was conducted with primers designed based on the sequence of bar gene, ubi promoter and target genes. Totally five plants transformed with the TaTBL gene and 6 plants transformed with the TaTST showed positive bands, which means exogenous genes have been integrated into host genomic DNA. Seedling leaves of transgenic plant were challenge inoculated with Erysiphe graminis f.sp. tritici conidispores and increased resistance was observed to varied degrees for different transformant.1.2 Analysis of wheat resistance-related genes by a transient expression systemTransient expression system was used to analyze the functions of six wheat resistance-related genes: TaTBL(Beclinl-like gene), TaPK1(serine/threonine protein kinase gene), TaTST(thiosulfate sulfurtransferase) Glb3s2( β -l,3-glucosidase) Cht4(chitinase gene) and Glb3s6(β -1,3-glucosidase) which had an increased expression against induction by Erysiphe graminis f.sp. tritici in other research. Target genes were constructed into plant expression vectors and transformed into leaf epidermal cells of powdery mildew-susceptible wheat variety by particle bombardment. GUS gene was co-transformed with either target gene to mark transformed cells. After transformation, leaf surface was inoculated with Erysiphe graminis f.sp. tritici conidiospores. 48 hours after inoculation, penetration of the fungus and formation of haustoria in transformed cells were observed to evaluate the effects of the target gene’s products on the invasion of powdery mildew. The results implied that five of these six genes, TaTBL, TaPKl, TaTST, Glb3s2 and Cht4, when transiently expressed in leaf epidermal cells of susceptible wheat variety, can partially inhibit penetration of conidios更多还原