'陕麦139'受条锈菌抑制表达相关基因的分析

以‘条中32’接种前后的小麦抗条锈病种质‘陕麦139’幼苗叶片为材料,利用抑制消减杂交技术,构建了条锈菌接种48 h小麦抗病品种叶片的抑制表达SSH-cDNA文库,通过测序以了解条锈菌侵染后被抑制表达的相关基因。从构建的文库中随机选取48个阳性克隆,进行测序,经过序列拼接等,获得高质量EST序列34条(Gen-Bank序列号为EL930132-EL930165),序列比对分析表明其中31条EST序列与已知功能的基因序列同源性较高,主要涉及植物的能量代谢途径、膜转运、信号传导及核酸加工等方面的基因,包括光合系统Ⅱ抗性蛋白D1、碳酸酵素、线粒体磷酸盐转运子、苹果酸脱氢酶、过敏性诱导反应蛋白和rRNA核酸内切酶等相关基因。此外,获得未知功能的EST序列3条。研究表明,小麦在条锈病菌侵染初期,许多基因的正常表达调控受到影响,如能量代谢和寄主细胞结构木质化,阻止小麦抗病过敏性反应等。     

铝胁迫下柱花草SSH文库构建及表达序列标签分析

柱花草栽培种热研2号（Stylosanthes guianensis‘Reyan2’）对铝毒有较强的耐受性。为了鉴定其在铝胁迫下的诱导基因,利用抑制消减杂交（SSH）技术构建在300μmol·L-1铝胁迫下正向cDNA文库。挑选插入片段大于300bp的600个克隆进行测序,共获得504条表达序列标签（EST）。序列重复性分析表明,其中12.1%的EST只有1次重复,61.4%的EST有2-16次重复,重复出现次数较高的EST是细胞色素P450（53次,占10.5%）、病原诱导型胰蛋白酶抑制剂（44次,占8.7%）和衰老相关蛋白（37次,占7.3%）。BLASTX分析显示,504条EST中有97种非冗余基因,其中包括46条功能已知基因和51条功能未知序列。46条功能已知EST中有30个为已报道铝胁迫相关基因,16个是新发现的铝胁迫相关基因。SSHcDNA文库提供的信息为阐明柱花草耐铝毒的分子机制提供了重要线索。     

白桦雌花序抑制性消减文库构建及EST分析

为研究白桦雌花序发育过程中特异基因的表达,以白桦雌花序样品为tester,雄花序样品为driver,利用SMART策略构建了白桦雌花序抑制性消减（SSH）文库。构建SSH文库的重组率为72%,插入片段的平均长度为400 bp左右。随机挑选文库克隆测序,获得150条EST序列,这些序列被GenBank的dbEST数据库收录,收录号为EE284580-EE284681,EE595316-EE595363。通过BlastX对EST进行功能注释,并对其中同源性较高的111条EST按功能进行分类,EST功能涉及了代谢、细胞防御、转录调节、能量代谢及信号传导等途径。发现了多个已知的控制花发育相关的EST,它们占已知功能EST的21%其功能涉及到调控花序形成和花分化、调控花粉与柱头亲和性以及调控花粉管发育等,包括MADS-box、S-locus F-box等基因。这些EST的获得为了解白桦花期基因表达,白桦花发育相关基因克隆和功能解析奠定了基础。     

条锈菌诱导的抗锈小麦种质的基因表达分析

以条锈菌接种前后的抗条锈病种质N 95175的小麦幼苗叶片为材料,利用抑制消减杂交技术,构建了条锈菌接种初期小麦抗性种质叶片的SSH-cDNA文库。通过对文库中随机选取的50个阳性克隆提取质粒,进行测序,共获得已知功能的EST序列14条,如蛋白激酶、锌指蛋白、细胞色素P 450和OM T 1等抗病相关基因,它们涉及植物的信号传导、转录调控、丙烷代谢途径及防卫反应等方面。     

苏麦3号感赤霉病近等基因系的选育及分子标记

小麦品种苏麦3号高抗由禾谷镰刀菌（Fusarium graminearum Schw.)引起的小麦赤霉病,已成为全世界广泛应用的抗源。为更精确地研究苏麦3号的赤霉病抗性,实验用感病品种川980为供体亲本,苏麦3号为轮回亲本,通过不断回交和自交,把川980的赤霉病感病基因导入苏麦3号,创造了感赤霉病的苏麦3号近等基因系（S016）,并得到以下结果：（1）育成的苏麦3号感赤霉病近等基因系,经两年多点抗赤霉病性状鉴定,表明该近等基因系感赤霉病感病性稳定,而其他形态特征,特性与苏麦3号一致。（2）用RAPD,RFLP分析抗,感苏麦3号近等基因系之间的差异,RFLP分析发现该近等基因系在2D染色体上存在差异。RPAD的随机扩增产物OPH191400可能与近等基因系中的赤霉病感病基因非连锁。（3）用近等基因系验证已报道的与小麦抗霉病基因有关的分子标记,均未发现这些分子标记与导致该近等基因系抗性差异的基因有关。     

小麦抗赤霉病品种NBS同源序列克隆与分析

根据二穗短柄草NBS-LRR类基因的保守序列设计同源引物,以小麦抗赤霉病品种苏麦3号、宁7840和望水白基因组DNA为模板,通过PCR扩增,得到43条序列,其中4条为非编码序列或结构域不完整;39条与植物抗病基因同源,其中的7条内部存在终止密码子,可能是假基因,经过比对分析,其余32条具有连续的开放阅读框和保守结构域,推导的氨基酸序列均具有Kinase-1a、Kinase-2和Kinase-3a及GLPL区等几个保守区,在GenBank中均能找到与之高度同源的其他物种的核酸序列,并且Kinase-2的最后一个氨基酸均为色氨酸(W),属于non-TIR类NBS基因。32条序列可分为4大类,它们之间核苷酸同源性为64%-98%,编码氨基酸同源性为22%-98%。根据序列分析随机设计5对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,7-1、s-3、s-4和w-2均能表达,说明这些片段可能是功能性抗病基因的部分序列;7-13不表达,再次证明属于假基因。32条序列在之前未被报道过,这些RGA可以作为筛选赤霉病功能性抗病基因的候选序列。     

白叶枯病菌胁迫下云南普通野生稻SSH文库的构建及抗病相关基因分析

以云南普通野生稻为材料,利用抑制差减杂交技术（SSH）,构建了白叶枯病菌胁迫的云南普通野生稻特异表达基因的差减文库.通过对文库所有阳性单克隆进行测序,聚类分析后共获得494条高质量的表达序列标签（EST）.经过BlastN分析,有417条与已知功能的序列有较高同源性;经BlastX分析,有104条EST与未知功能蛋白或假定蛋白有较高相似性,49条EST未能找到同源匹配,341条EST与已知功能蛋白有较高同源性.初步分析发现,这些基因主要涉及能量代谢、蛋白质代谢、核酸代谢、防御与抗逆应答反应、信号转导、光合作用及膜运输等代谢过程.使用半定量RT-PCR研究了7个可能与白叶枯病抗性相关的EST序列在云南普通野生稻对照和白叶枯病菌处理的叶片中的表达情况,并获得这些基因的表达谱.结果发现,克隆编号为OR7,OR68和OR826的EST受白叶枯病菌胁迫诱导上调表达,其中OR826 EST在蛋白数据库中无同源序列,可能是一类新的白叶枯病抗性基因,而组成型表达的OR143 EST在对照和接菌处理的叶片中均能检测到其mRNA的表达,但其表达量在白叶枯病菌胁迫48 h后逐渐增强,推测这些基因直接参与了云南普通野生稻抗病防御反应.本研究为从云南普通野生稻中发掘和克隆新的白叶枯病抗性基因提供了理论依据,为进一步研究云南普通野生稻抗白叶枯病的分子机制奠定了基础.     

小麦PAL基因的克隆及赤霉菌诱导下的表达分析

利用苯丙氨酸解氨酶(PAL,phenylalanine ammonia-lyase)基因保守区域从小麦抗赤霉病材料苏麦3号中克隆获得4个PAL基因,分别命名为Ta PAL1、Ta PAL2、Ta PAL3、Ta PAL4。4个基因的开放阅读框(ORF,open reading frame)长度分别为2142 bp、2016 bp、2118 bp和2139 bp,分别编码714个、672个、706个和713个氨基酸。基因序列比对发现其相似性达到88.35%,所编码的氨基酸相似性为91.92%,氨基酸序列分析表明4个基因都包含HAL-PAL结构域及PAL结构域。通过接种禾谷镰刀菌,利用荧光定量PCR对PAL基因进行表达分析发现,4个PAL基因全部为上调表达,其中Ta PAL2、Ta PAL3和Ta PAL4最为明显。PAL基因的上调表达,说明PAL基因在小麦抵抗赤霉病菌侵染的机制中可能起着重要作用。     

青枯菌诱导的花生基因表达谱SSH分析

以抗青枯病花生种质‘J4’和‘中花6号’、感青枯病花生品种‘中花12号’为材料,用强产青枯菌毒菌株(Ralstonia solanacearum)对其根系分别接种,采用抑制差减杂交(SSH)技术检测花生根系应答侵染的基因表达谱变化,并对文库中差异基因进行Real-time PCR分析。结果表明:经菌液PCR检测对挑选出的1 036阳性克隆片段进行测序及片段整合分析,获得162条花生基因,有功能注释的基因58条,其中44条基因参与了细胞结构(6%)、信号转导(12%)、抗病防御(5%)、转录调控(12%)等生理过程。用Real-time PCR技术对7个基因在‘中花6号’和‘中花12号’中的表达模式分析结果表明,6个基因在青枯菌侵染早期在抗病材料‘中花6号’中呈上调表达,可能与青枯病抗性直接相关。     

小麦种子中几丁质酶基因的扩增及序列分析

根据http://www.tigr.org中与小麦几丁质酶基因相关的序列TC187877,设计引物,分别从小麦品种Gamenya和苏麦3号中扩增到大小约为1 000bp的片段。经序列测定和软件分析比较,发现这些片段所编码的蛋白质氨基酸序列,都有CHITINASE-19.1和CHITINASE-19.2的基序,为第II类几丁质酶基因。扩增的核酸序列在GenBank上发表,登录号分别为AY973229和AY973230。     

Molecular characterization of Fusarium head blight resistance in Wangshuibai with simple sequence repeat and amplified fragment length polymorphism markers.

Molecular mapping of Fusarium head blight (FHB) resistance quantitative trait loci (QTL) and marker-assisted selection of these QTL will aid in the development of resistant cultivars. Most reported FHB resistance QTL are from 'Sumai 3' and its derivatives. 'Wangshuibai' is a FHB-resistant landrace that originated from China and is not known to be related to 'Sumai 3'. A mapping population of 139 F(5:6) recombinant inbred lines was developed from a cross of 'Wangshuibai' and 'Wheaton'. This population was developed to map the FHB-resistant QTL in 'Wangshuibai' and was evaluated twice for Type II FHB resistance. A total of 1196 simple sequence repeat and amplified fragment length polymorphism markers were screened on this population, and four FHB resistance QTL were detected. A major QTL near the end of 3BS explained 37.3% of the phenotypic variation. Another QTL on 3BS, located close to the centromere, explained 7.4% of the phenotypic variation. Two additional QTL on 7AL and 1BL explained 9.8% and 11.9% of the phenotypic variation, respectively. The simple sequence repeat and amplified fragment length polymorphism markers closely linked to these QTL may be useful for stacking QTL from 'Wangshuibai' and other sources to develop cultivars with transgressive FHB resistance.     

Targeted molecular mapping of a major wheat QTL for Fusarium head blight resistance using wheat ESTs and synteny with rice.

A major QTL for resistance to Fusarium head blight (FHB) in wheat, Qfhs.ndsu-3BS, has been identified and verified by several research groups. The objective of this study was to increase the marker density in this QTL region using STS (sequence-tagged site) markers developed from wheat expressed sequence tags (ESTs) near Qfhs.ndsu-3BS. Because wheat chromosome 3BS and rice chromosome 1S are syntenous, the sequences of P1-derived artificial chromosome (PAC) and (or) bacterial artificial chromosome (BAC) clones covering the sub-distal portion of rice chromosome 1S were used as queries for a BLASTn search to identify wheat ESTs most likely near Qfhs.ndsu-3BS. Sixty-eight out of 79 STS primer pairs designed from wheat ESTs amplified PCR products from the genomic DNA of Triticum aestivum 'Chinese Spring'. Twenty-eight STS markers were localized on chromosome 3BS by aneuploid analysis. Six out of the nine STS markers that could be mapped in the T. aestivum 'Sumai 3'/T. aestivum 'Stoa' population had higher R2 and LOD values for this QTL than the most significant marker reported previously. Therefore, leveraging genome sequence information available in rice for wheat genetics is an effective strategy to develop DNA markers for Qfhs.ndsu-3BS, and this strategy may have broad applications for targeted mapping of other traits in cereal crops.     

Single-strand conformational polymorphism markers associated with a major QTL for fusarium head blight resistance in wheat

A major quantitative trait locus (QTL) associated with resistance to Fusarium head blight (FHB) was identified on chromosome 3BS between simple sequence repeat (SSR) markers Xgwm389 and Xgwm493 in wheat 'Ning 7840', a derivative from 'Sumai 3'. However, the marker density of SSR in the QTL region was much lower than that required for marker-assisted selection (MAS) and map-based cloning. The objective of this study was to exploit new markers to increase marker density in this QTL region by using single-strand conformational polymorphism (SSCP) markers developed from wheat expressed sequence tags (ESTs) on 3BS bin 8-0.78-1.0. Sixty-nine out of 85 SSCP primer pairs amplified PCR (polymerase chain reaction) products from the genomic DNA of 'Chinese Spring'. Thirty-four primer pairs amplified PCR products that could form clear ssDNA (single strand DNA) bands through denaturation treatment. Ten SSCP markers had polymorphisms between 'Ning 7840' and 'Clark'. Five of the ten polymorphic SSCP markers were located on chromosome 3B by nulli-tetrasomic analysis. Three SSCP markers (Xsscp6, Xsscp20, and Xsscp21) were mapped into the region between Xgwm493 and Xgwm533, and possessed higher coefficient of determination (R2) than Xgwm493 and Xgwm533. The SSCP markers, Xsscp6, Xsscp20, and Xsscp21, can be used for map-based cloning of the QTL and for marker-assisted selection in FHB resistance breeding.     

Validation of EST-derived STS markers localized on Qfhs.ndsu-3BS for Fusarium head blight resistance in wheat using a 'Wangshuibai' derived population

A few EST-derived STS markers localized on Qfhs.ndsu-3BS, a major QTL for resistance to Fusarium head blight (FHB) in wheat, have been previously identified in the 'Sumai 3'/'Stoa' population. In this study, we used a 'Wangshuibai' (resistant)/'Seri82' (susceptible) derived population, linkage group, QTL, and quantitative gene expression analysis to assess the genetic background dependence and stability of the EST-derived STS markers for use in marker aided selection to improve FHB resistance in wheat. Based on our results, a QTL in the map interval of Xsts3B-138_1-Xgwm493 on chromosome 3BS was detected for FHB resistance, which accounted for up to 16% of the phenotypic variation. BLASTN analysis indicated that Xsts3B-138_1 sequence had significant similarity with the resistance gene analogue. Real-time quantitative PCR showed that the relative expression of Xsts3B-1381 in 'Wangshuibai' at 96 h after inoculation was 2.6 times higher than 'Seri82'. Our results underlined that EST-derived STS3B-138 markers could be predominantly used in marker aided selection to improve FHB resistance in wheat.     

A resistance-like gene identified by EST mapping and its association with a QTL controlling Fusarium head blight infection on wheat chromosome 3BS.

Fusarium head blight (FHB) is a major disease in the wheat growing regions of the world. A quantitative trait locus (QTL) on the short arm of chromosome 3B controls much of the variation for resistance. The cloning of candidate disease-resistance genes for FHB QTLs on chromosome 3B can provide further elucidation of the mechanisms that control resistance. However, rearrangements and divergence during plant genome evolution often hampers the identification of sequences with similarity to known disease-resistance genes. This study focuses on the use of wheat expressed sequence tags (ESTs) that map to the region on chromosome 3B containing the QTL for FHB resistance and low-stringency BLAST searching to identify sequences with similarity to known disease-resistance genes. One EST rich with leucine repeats and low similarity to a protein kinase domain of the barley Rpg1 gene was identified. Genetic mapping using a Ning894037 x Alondra recombinant inbred (RI) population showed that this EST mapped to the QTL on the short arm of chromosome 3B and may represent a portion of a newly diverged gene contributing to FHB resistance. The EST is a new marker suitable for marker-assisted selection and provides a starting point to begin map-based cloning for chromosome walking and investigate new diverged genes at this locus.