【作者】 马璐琳；

【导师】 刘大钧；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 小麦赤霉病(Fusarium head blight, FHB)是一种由镰刀菌(Fusarium graminearum)引起的,严重危害小禾谷类和玉米等作物的真菌性病害。赤霉病不仅会造成作物严重减产,降低种子品质,而且赤霉病菌还会在籽粒中聚积一些单端孢霉烯族(trichothecenes)毒素,严重危害人畜健康。其中脱氧雪腐镰刀菌烯醇(Deoxynivalenol, DON)是最重要的一种毒素,不仅危害人畜健康,而且还被认为是促进赤霉病发病的毒性因子。培育抗赤霉病特别是抗DON积累的小麦品种是减轻赤霉病危害的有效途径。目前,在赤霉病五种抗性类型中对TypeⅠ(抗侵染)和TypeⅡ(抗扩展)的研究较多且深入。随着近年来愈来愈重视食品安全问题,毒素积累抗性类型(TypeⅢ)已经引起广泛关注,但相关研究报道仍然较少,因此迫切需要克隆小麦毒素抗性相关基因并系统深入地研究毒素抗性机制。本研究利用Affymetrix小麦基因芯片对经DON诱导后抗赤霉病小麦品种望水白穗组织的基因表达谱进行分析,发现了多个受DON诱导后差异表达的基因。初步分析表明,具有推断功能的表达显著上调基因中,许多基因与抗病(菌)相关,如：谷胱甘酞转移酶、苯丙氨酸解氨酶、细胞色素P450酶以及抗病蛋白等,尤其还发现了一类与已报道的拟南芥中降解DON的DOGT1基因同一家族的尿核苷二磷酸葡萄搪基转移酶(UDP-glucosyltransferase, UGTs)基因。为了进一步验证芯片杂交结果的可靠性并研究这些基因受DON诱导后的表达模式,本研究在这些可能的抗病(菌)有关基因中,选取了部分基因进行半定量RT-PCR分析,结果发现它们都受DON诱导上调表达,表达情况与芯片结果一致,推测这些基因可能与望水白抗DON有关。本研究还进一步证明基因芯片是大规模分析小麦对DON反应的基因表达谱高效而准确的手段。芯片杂交结果为进一步研究小麦对赤霉病(或DON)的抗性以及抗赤霉病(或DON)相关基因的筛选与克隆提供了有价值的信息。芯片杂交结果发现,在上调表达基因中基因编号为CA695961的一个EST上调表达25.5倍,该EST序列与已报道的与抗DON有关的UGTs家族基因同源,本研究根据该EST碱基序列设计基因特异引物,筛选本实验室构建的DON诱导望水白穗组织cDNA文库,从中筛选到一个全长的小麦UGT基因TaUGT3 (GenBank accession FJ236328).该基因的cDNA全长1,755bp,包含编码一个496氨基酸的UGT基因的ORF框。TaUGT3与已报道的来自于拟南芥中能降解DON的DOGT1基因在氨基酸水平上有较高的同源性(43%)。半定量RT-PCR结果显示TaUGT3基因受DON的诱导上调表达,并且在抗赤霉病小麦品种望水白不同组织的表达情况有所差别,根与叶部的表达高峰相对穗部推迟了12h。虽然TaUGT3在抗、感赤霉病小麦品种穗中的表达趋势基本一致,但其在抗病品种望水白中最高峰时的表达强度明显超过感病品种绵阳85-45。利用中国春缺体-四体和缺失系系列,将TaUGT3基因的3个直向同源基因分别定位于普通小麦第三部分同源群染色体的短臂上。洋葱表皮亚细胞定位结果显示,TaUGT3基因主要位于细胞膜上和细胞核中。通过农杆菌介导法将TaUGT3基因转化拟南芥,获得阳性转基因植株。Northern杂交显示,转基因植株中TaUGT3基因可以超表达,且不同植株中表达水平有较大差异。DON抗性鉴定表明阳性转基因植株中TaUGT3基因过量表达在一定程度上可以提高拟南芥对DON的耐受力,推测TaUGT3基因可能在小麦抗DON反应中发挥作用。更多还原

【Abstract】 Wheat Fusarium head blight (FHB) caused by Fusarium graminearum, also called scab, is a destructive fungus disease of small grain cereals and maize. Scab can not only causes yield loss, more seriously is that it can also deteriorate seed quality by contaminating the infected grains with trichothecenes toxins, which are harmful to both human and animal health. Deoxynivalenol (DON) is one of the most important toxin members. It was proposed that DON acted first as a virulence factor during fungal pathogenesis and then accumulated in grain to levels posing a threat to human and animal health. To date, among the five types of FHB resistance, Type I (resistance to the initial infection) and Type II (resistance to the spread of infection) have been intensively studied. Recently, with the increasing attentions on food safety, more and more studies on TypeⅢ(resitance for toxin accumulation) were initiated. However, very few research of TypeⅢresitance were reported. Therefore, it is of great significance to identify the DON resistance-related genes and characterize the underlying molecular mechanism of DON resistance of wheat.In the present research, by expression analysis of DON-induced samples using GeneChip(?) Wheat Genome Array(http://www.affymetrix.com/products/arrays/specific /wheat.affx), a DON-resistance related gene TaUGT3 (GenBank accession FJ236328) were cloned and characterized from a scab resistant wheat(Triticum aestivum L.) variety Wangshuibai. The full-length cDNA of TaUGT3 was 1,755 bp and contained a putative open reading frame (ORF) with 496 amino acids encoding a UDP-glucosyltransferase (UGT). TaUGT3 showed high similarity in amino acid level with DOGT1 gene in Arabidopsis, which has been confirmed its function of detoxification of DON. TaUGT3 was located on the group 3 chromosomes of wheat using nullitetrasomic lines and deletion lines of ChineseSpring. Co-transformation of TaUGT3 with GFP genes to onion epidermic cells using transient transformation technique by microprojectile bombardment indicated the subcellular location of the protein encoded by TaUGT3 was in the plasma membrane and nuclear. Transformation and overexpression of the TaUGT3 gene in Arabidopsis could enhance tolerance against DON.更多还原