【作者】 尚毅；

【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】 脱氧雪腐镰刀菌烯醇(deoxynivalenol,简称DON)在小麦赤霉病(Fusarium head blight,FHB)发病过程中起重要作用,是一种毒性因子。禾谷镰刀菌的侵染能力依赖于其产生DON的能力,DON单独处理即可引起较典型的赤霉病症状,抗病品种能显著地降低病穗组织中DON的含量,因而具有不同FHB抗性的品种其籽粒中DON含量也有很大的差别。现在已经发现了一些和抗毒素或降解毒素积累相关的基因,但由于小麦抗DON是个复杂的数量性状,而且小麦抗赤霉病和抗DON的机理还不清楚,所以从小麦中鉴定一些新的对DON反应密切相关的基因显得尤为重要。本研究以高抗赤霉病小麦品种望水白为材料,利用SMART技术构建了DON诱导的小麦穗组织λ噬菌体cDNA文库,旨在揭示DON诱导后小麦基因的表达特点并克隆相关的基因。未扩增文库的滴度大约为3×106,扩增后文库的滴度大约为2×109,平均插入片段1053bp,文库以19个混合池保存。改进了基于PCR的噬菌体cDNA文库筛选方法,用液体分装的方法,替代了的文库筛选的关键步骤—涂板分区,省去了噬菌体文库铺平板、浸染、培养、划块洗脱的操作过程,使筛库的工作量减少,速度和效率提高。利用Affymetrix小麦基因组芯片对高抗赤霉病小麦品种望水白经DON诱导后穗组织基因表达特点进行了分析,结果发现,一个编码PDR型转运蛋白的EST受DON诱导后上调表达45倍。根据该EST设计引物筛选小麦基因组TAC文库,得到一个包含该基因的TAC单克隆。利用染色体walking对该单克隆测序,用Softberry软件进行基因预测,根据预测基因的5’和3’非翻译区设计引物,从DON诱导的小麦望水白穗组织cDNA中克隆出该转运蛋白基因。该基因基因组全长7377bp,包含19个外显子,CDS长度为4308bp,编码1435aa、分子量161kDa的蛋白质,蛋白序列比对表明该蛋白属于PDR蛋白家族,将该基因命名为TaPDR1(Triticum asetivum Pleiotropic Drug Resistance)。利用一套中国春缺体-四体系将TaPDR1基因定位在小麦5A染色体上。半定量RT-PCR表明TaPDR1在望水白穗中受DON和禾谷镰刀菌诱导表达,表明其参与了植物抗病防御反应。该基因在望水白感病突变体中低水平表达进一步证明TaPDR1与赤霉病抗性有关。TaPDR1的表达不受与生物胁迫相关的激素如JA和SA、非生物胁迫因子如热、冷、伤害和NaCl的诱导。但TaPDR1受到Al3+和游离Ca2+的诱导表达,推测[ca2+]i介导了TaPDR1的表达信号。进一步构建了双链RNA干扰载体,通过农杆菌介导转化小麦,初步研究了该基因的功能。二穗短柄草(短柄草)是一种禾本科新的模式植物,本研究开发了一个可以在小麦和短柄草中通用的PDR1基因特异引物TR2。从23份短柄草中克隆出39个PDR1同源基因片段,通过对这些基因序列的比对分析,发现PDR1基因在短柄草中高度保守,同源性在95%-99%。利用在PDR1基因中找到的SNP位点设计了两个CAPS标记,可以区分来自于短柄草不同染色体组的PDR1基因的两个拷贝。利用这两个CAPS标记可以将这些同源基因分成两种类型E型和H型,系统分析表明每一种类型形成一个组,支持率很高,推测六倍体短柄草中的PDR1基因的2个拷贝分别来自于2个染色体组,试验中的二倍体可能就是六倍体基因组中的一个染色体组的供体。PDR1基因的两个拷贝在多倍体中的进化速度不同,显示这两种类型在年代上的差异,或两种类型的突变率或受到的选择压不同,系统分析同样表明,六倍体短柄草中的E型PDR1基因有一个共同的染色体组起源,而六倍体短柄草中的H型PDR1基因是多起源的。以上结果反映了短柄草复杂的多倍体进化历史。利用这两类PDR1基因片段的合并序列,重建了六倍体短柄草的系统进化树。更多还原

【Abstract】 DON (deoxynivalenol), a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. Wheat tissue showed typical FHB symptom when treated with DON only. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. At present, several genes related to DON resistance have been isolated from various plants. In order to better understand the molecular mechanisms of FHB or DON resistance, it is critical to identify new genes that are expressed closely related to DON treatment, because the inheritance of FHB resistance and DON resistance is a complicated quantitative trait and the mechanisms under FHB resistance have not yet been clarified.In order to reveal the gene expression patterns of wheat induced by DON and clone genes related to DON resistance, a cDNA library of spikes of scab-resistant wheat Wangshuibai induced by DON was constructed with SMART cDNA library construction kit. The titer of the primary library was 3×106pfu/mL, and the titer of the amplified library reached at 2×109 pfu/mL. The inserted fragments size of the positive clones was 1053 bp at average. The library was stored in 19 mixing pools. In the present reseach, we improved the PCR-based procedure of screening phage cDNA library. The key step in screening library, ’plate-division’ was replaced by’partition in liquid’, which avoids tedious plating, infection and washing procedures. This modification not only decreased the workload of library screening, but also improved the speed and efficiency of obtaining the positive clones.In this study, GeneChip analysis indicated that an EST encoding an ABC (ATP-Binding Cassette) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers was designed based on the EST sequence, and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kDa. BLAST analysis indicated that the gene may belong to PDR (pleiotropic drug resistance) sub-family, and hence designated as TaPDRl (Triticum asetivum Pleiotropic Drug Resistance). TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDRl was up-regulated by both DON and F. graminearum. Expression patterns of TaPDR1 were different in wildtype Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCl. However, TaPDRl expression was regulated by Al3+ and [Ca2+], indicating that [Ca2+]i might mediate the signal of TaPDR1 expression. A double stranded RNA interference vector was constructed. Preliminary function of the gene was studied by Agrobacterium mediated transformation method into wheat.A pair of primers, TR2, was designed based on the sequence of TaPDR1 and used for amplification in 23 ecotypes of Brachypodium distachyon from different geographical regions. Detailed sequence analysis showed that the PDR1 genes were highly conserved in B. distachyon. The sequence similarities from different B. distachyon species were more than 95%. Two CAPS markers, which were designed based on SNP sites found in the PDR1 gene of B. distachyon, could differentiate the PDR1 homologous genes from different B. distachyon genome. Based on restriction site analysis, the PDR1s were classified as E type or H type. From 23 B. distachyon ecotypes,39 PDR1s were identified. All ecotypes had either 1 or 2 PDR1 copies. All but one diploid and tetraploid ecotype had only a single H type PDR1. All but one hexaploid ecotype had both an E type and an H type PDR1. Phylogenetic analysis revealed that each type formed a well-supported cluster. The two PDR1 types appeared to evolve differently. These different evolutionary patterns could indicate a difference in age between the two types or might indicate different mutation rates or selective pressures on the two types. The phylogenetic analysis also revealed that the hexaploid ecotypes shared a genomic origin for their E type PDR1,but there were multiple origins for hexaploid H type PDR1.Overall, the results suggest that tetraploid and hexaploid might be misnomers in B. distachyon and suggest a complex polyploidization history during B. distachyon evolution.更多还原