谷子抗病基因同源序列的克隆与分析

谷子锈病是谷子上的一种流行性强、毁灭性大的病害,严重影响谷子生产.种植抗病品种是防治锈病最经济有效的方法,但谷子抗锈病种质资源非 常贫乏,而且高抗锈病的材料其农艺性状又很差.很难通过传统育种方法培育抗锈病品种,因此克隆谷子抗锈病基因尤为重要.目前克隆到的许多植 物抗病基因编码的氨基酸序列都有一定的保守结构域.根据抗病基因保守结构域,已克隆的抗病基因主要分为5类,其中最主要的是NBS-LRR(nu- leotide-binding site leucine-rich epeat)和STK(Se-rine／Threonine protein kinase)类.因而根据抗病基因保守结构域设计引物,从植物的DNA中扩增植物的抗病基因同源序列RGA(resistance geneanalogs)更加快捷有效,目前通过RGA方法克隆植物抗病基因已有报道[1,2].     

Identifying resistance gene analogs associated with resistances to different pathogens in common bean

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.     

柑橘黄龙病侵染相关抗病基因同源序列的克隆鉴定与表达分析

[目的]从黄龙病耐病寄主植物cDNA中筛选抗病基因相关序列并对其进行表达分析研究。[方法]根据已克隆的植物抗性基因表达产物NBS-LRR保守区域设计简并引物,以耐HLB的柑橘属柚cDNA为模板扩增RGAs,并进行实时荧光定量PCR。[结果]通过RFLP分析及克隆测序共得到5个NBS类抗病基因相似序列(RGAs)片段,在GenBank上登录号为HM777043～HM777047。通过Clustalx、DNAMAN等软件分析5个RGAs及其推导的氨基酸的相似性,结果显示它们均含有典型NBS-LRR类抗性基因所具有的保守区域:P-loop、Kinase-2a、GL-PLAL,其与已克隆的烟草N、亚麻L6、拟南芥RPS2、RPS5、RPP8、RPM1等抗病基因在保守区域氨基酸水平上的相似性为19.71%～42.86%。根据得到的序列设计特异性引物,对5个RGAs在HLB侵染过程中的表达进行定量PCR,结果显示嫁接病芽接穗后的8次连续采样中5个RGA的表达受到不同程度的调控。[结论]表明5个RGAs可能与黄龙病的侵染有关。     

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.)

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. RESULTS: Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. CONCLUSION: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.     

Identification and Characterization of Random Amplified Polymorphic DNA Markers Linked to a Major Gene (Cr2) for Resistance to Cronartium ribicola in Pinus monticola

ABSTRACT DNA markers tightly linked to resistance (R) genes provide a very powerful tool for both marker-assisted selection in plant breeding and positional cloning of R genes. In the present study, a linkage of random amplified polymorphic DNA (RAPD) markers to the single dominant gene (Cr2) for resistance to white pine blister rust fungus (Cronartium ribicola) was investigated in western white pine (Pinus monticola). A mapping population of 128 individual megagametophytes was generated from seeds of a heterozygous resistant tree (Cr2/cr2), and the corresponding seedlings of each megagametophyte were subjected to the test of phenotype segregation by inoculation with C. ribicola. Bulked segregant analysis and haploid segregation analysis identified eight robust RAPD markers linked to Cr2. This constitutes the first Cr2 genetic linkage map spanning 84.7 cM with four markers only 3.2 cM from Cr2. One sequence (U256-1385) of these linked markers was significantly similar to the Ty3/gypsy-like long terminal direct repeats retrotransposons. Another marker, U570-843, had no significant similarity to any entry in either GenBank or the loblolly genomics data bank. As presumed that the average physical distance per centimorgan is about 10 Mb in P. monticola, it is probably unrealistic to use these DNA markers for positional cloning of the Cr2 gene.     

番茄黄萎病抗病基因Ve的AFLP和SSR分子标记

 本研究以番茄抗病品种05046与感病品种051355配制杂交组合,接种鉴定F1代及F2代分离群体的黄萎病发生情况,结果表明,番茄黄萎病属单基因显性遗传。用545对AFLP引物和101对SSR引物对两个亲本、抗感池及F2代分离群体进行AFLP和SSR分析,得到3个与番茄抗黄萎病基因Ve连锁的AFLP标记和1个SSR标记,分别是E66M84-A、E78M84-D、E66M40-A和SSR599,与抗病基因Ve的连锁遗传距离分别为10.3、14.2、30.5 和12.5 cM。     

Cloning and characterization of NBS-LRR encoding resistance gene candidates from Tomato Leaf Curl New Delhi Virus resistant genotype of Luffa cylindrica Roem

We employed degenerate primers to amplify nucleotide-binding site (NBS) domain of resistance gene candidates (RGCs) from Tomato Leaf Curl New Delhi Virus (ToLCNDV) resistant Luffa cylindrica (sponge gourd) genotype, DSG-6. Sixteen non-redundant sequences of RGCs were identified with un-interrupted open reading frames (ORFs) and high amino acid sequence homologies (60–98%) to various nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins from GenBank database. Alignment of the deduced amino acid sequence and phylogenetic analysis of the NBS domain revealed six and ten sponge gourd (sg) RGCs belong to the Toll Interleukin Receptor (TIR) and non-TIR group of NBS-LRR genes, respectively. The sgRGCs consisted of conserved NB-ARC [homologous region shared with APAF-1 (apoptotic protease-activating factor-1), R proteins and CED-4 (Caenorhabditis elegans death-4 protein)] domain from P-loop nucleoside triphosphatase (NTPase) family and characteristic P-loop, Kinase-2, RNBS-A, Kinase-3A and GLPL motifs. The comparative analysis of expression profiles of sgRGCs in asymptomatic and field-driven symptomatic leaf tissues of ToLCNDV resistant and susceptible genotypes revealed RGCLc28 is expressed consistently in resistant genotypes. The differentially expressed RGCLc28 of DSG-6 is predicted to have strong association with the resistance trait against the leaf curl and mosaic disease in sponge gourd and may serve as important genomic resource for candidate gene discovery.     

New gene for bacterial blight resistance in rice located on chromosome 12 identified from minghui 63, an elite restorer line

ABSTRACT Bacterial blight, caused by Xanthomonas oryzae pv. oryzae, is a serious disease of rice worldwide. A new dominant gene for bacterial blight resistance in rice, Xa25(t), was identified from Minghui 63, a restorer line for a number of rice hybrids that are widely cultivated in China. This gene conferred resistance to Philippine race 9 (PXO339) of X. oryzae pv. oryzae in both seedling and adult stages. It was mapped to the centromeric region of chromosome 12, 2.5 cM from a disease resistance gene-homologous sequence, NBS109, and 7.3 cM from a restriction fragment length polymorphism marker, G1314. The genomic location of this gene is similar to the previously identified blast resistance genes, Pi-ta and Pi-ta2.     

Identification and Molecular Mapping of a Gene in Wheat Conferring Resistance to Mycosphaerella graminicola

ABSTRACT Septoria tritici leaf blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici), is an economically important disease of wheat. Breeding for resistance to STB is the most effective means to control this disease and can be facilitated through the use of molecular markers. However, molecular markers linked to most genes for resistance to STB are not yet available. This study was conducted to test for resistance in the parents of a standard wheat mapping population and to map any resistance genes identified. The population consisted of 130 F(10) recombinant-inbred lines (RILs) from a cross between the synthetic hexaploid wheat W7984 and cv. Opata 85. Genetic analysis indicated that a single major gene controls resistance to M. graminicola in this population. This putative resistance gene is now designated Stb8 and was mapped with respect to amplified fragment length polymorphism (AFLP) and microsatellite markers. An AFLP marker, EcoRI-ACG/MseI-CAG5, was linked in repulsion with the resistance gene at a distance of approximately 5.3 centimorgans (cM). Two flanking microsatellite markers, Xgwm146 and Xgwm577, were linked to the Stb8 gene on the long arm of wheat chromosome 7B at distances of 3.5 and 5.3 cM, respectively. The microsatellite markers identified in this study have potential for use in marker-assisted selection in breeding programs and for pyramiding of Stb8 with other genes for resistance to M. graminicola in wheat.     

甜菜NBS-LRR家族基因的鉴定与分析

为明确甜菜中由抗性基因R编码的包含富亮氨酸重复序列（leucine-rich repeat,LRR）和核苷酸结合位点（nucleotide-binding site,NBS）的家族成员及其功能,基于甜菜基因组全长序列,利用HMMER、TBtools、Pfam、NCBI等软件和在线程序对甜菜NBS-LRR家族成员进行筛选和鉴定,采用生物信息学方法对鉴定到的成员进行亚家族分类、染色体定位、结构域分析、进化树构建、顺式元件分析和同源序列筛选。结果显示,从甜菜基因组中最终筛选鉴定到267条NBS-LRR家族基因序列,占甜菜基因组的0.614%,通过对267条基因序列进行结构域预测并进行分类,分属于N型、NL型、CNL型、TNL型和RNL型5个亚家族,分别包含110、25、128、3和1条序列。甜菜NBS-LRR家族基因大多位于2号、3号、4号和7号染色体上,根据基因簇划分原则发现有73.25%的基因以基因簇形式存在。经Clustal Omega和MEME在线程序对CNL型亚家族中具有完整卷曲螺旋（coiled-coil,CC）、NBS和LRR结构域的24条基因序列进行结构域保守性分析,共发现7个保守性较高的基序,基于CNL型亚家族128条基因序列构建的进化树显示CNL型亚家族的系统进化受CC、NBS和LRR结构域的影响较大。甜菜NBS-LRR家族基因含有大量植物激素相关顺式元件和多种胁迫响应元件,部分序列含有植物生理响应元件。甜菜NBS-LRR家族基因与菠菜和藜麦的抗病蛋白同源性较高。     

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.     

Isolation of Molecular Markers Linked to the <Emphasis Type="Italic">Cry</Emphasis> Locus Conferring Resistance to <Emphasis Type="Italic">Cucumber mosaic cucumovirus </Emphasis>Infection in Cowpea

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F₂ cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively. Received 16 December 1999/ Accepted in revised form 22 March 2000     

草地螟触角普通气味结合蛋白基因的克隆及序列分析

利用RT-PCR结合RACE技术克隆了编码草地螟(Loxostege sticticalis)触角中一种普通气味结合蛋白的基因。序列分析表明,成熟蛋白的序列中含有阅读框序列,推导的氨基酸序列与已报道的11种鳞翅目昆虫普通气味结合蛋白Ⅰ比较,平均相似度在62.3%左右,并拥有气味结合蛋白的典型结构特征。该基因归属于普通气味结合蛋白Ⅰ基因亚族。因此将它命名为LstiGOBP1。     

Incidence of thiophanate-methyl resistance in Cercospora kikuchii within a single lineage based on amplified fragment length polymorphisms in Japan

We collected 247 isolates of Cercospora kikuchii from soybean seeds with typical purple stain symptoms from 15 prefectures in Japan. Of the 247 isolates, 93 were sensitive to thiophanate-methyl, a benzimidazole used to control this soybean disease; the remaining 154 were highly resistant to the fungicide. To examine genetic variability among the population of 247 isolates, we developed amplified fragment length polymorphism (AFLP) markers. An AFLP primer pair generated DNA fingerprint polymorphisms among the sample isolates, and with the unweighted pair-grouping method to cluster arithmetic means of the similarity coefficients among all pairs of the fingerprint patterns, the isolates were divided into four lineages (I to IV). Of the 247 isolates, 225 belonged to lineage I, including all isolates that were resistant to thiophanate-methyl. To determine whether the resistance of these isolates was related to mutations in the β-tubulin gene, we amplified partial nucleotide sequences of the gene from 29 representative isolates, including 12 that were resistant to thiophanate-methyl, by means of the polymerase chain reaction. The resistant isolates had identical nucleotide sequence with a one-step change at codon 198, in which the amino acid glutamic acid had been replaced by alanine. The evidence thus suggests that thiophanate-methyl resistance might have arisen in lineage I, the largest of the four lineages. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB214511 to AB214515     

Genetic specificity in the white pine-blister rust pathosystem

ABSTRACT Four of eight white pine species native to western North America surveyed for resistance to white pine blister rust by artificial inoculation showed classical hypersensitive reactions (HR) at frequencies ranging from very low to moderate. Mendelian segregation, indicating a single dominant allele for resistance (Cr3), was observed in southwestern white pine (Pinus strobiformis), as it was previously in sugar pine (P. lambertiana, Cr1) and western white pine (P. monticola, Cr2). HR was present at a relatively high frequency (19%) in one of five bulk seed lot sources of limber pine (P. flexilis), and was also presumed to be conditioned by a single gene locus, by analogy with the other three species. HR was not found in whitebark pine (P. albcaulis), Mexican white pine (P. ayacahuite), foxtail pine (P. balfouriana), or Great Basin bristlecone pine (P. longaeva), but population and sample sizes in these species may have been below the level of detection of alleles in low frequency. When challenged by (haploid) inocula from specific locations known to harbor virulence to Cr1 or Cr2, genotypes carrying these alleles and Cr3 reacted differentially, such that inoculum virulent to Cr1 was avirulent to Cr2, and inoculum virulent to Cr2 was avirulent to Cr1. Neither of these two inocula was capable of neutralizing Cr3. Although blister rust traditionally is considered an exotic disease in North America, these results, typical of classic gene-for-gene interactions, suggest that genetic memory of similar encounters in past epochs has been retained in this pathosystem.     

Origin and distribution of cr2, a gene for resistance to white pine blister rust in natural populations of Western white pine

ABSTRACT The distribution and frequency of the Cr2 gene for resistance to white pine blister rust (Cronartium ribicola) in western white pine (Pinus monticola) was surveyed in natural populations of the host by inoculation of open-pollinated seedlings from 687 individual seed parents from throughout most of the species' range. Because Cr2 is dominant and results in a conspicuous hypersensitive reaction (HR) in pine needles, the phenotype can readily be detected in offspring of susceptible seed parents fertilized by unknown Cr2 donors in the ambient pollen cloud. Gametic frequencies of Cr2 were thus determined as the proportion of total challenged seedlings that were pollen receptors exhibiting the Cr2 phenotype. Zygotic frequencies, the proportion of seed parents with progeny that segregated in Mendelian ratios for the Cr2 phenotype to the total number of parents, were a complementary, though less precise, measure. Cr2 frequency was rare overall, ranging from 0.004 to 0.008 in the Sierra Nevada to about 0.001 in the central Cascade Range; it was undetectable further north in the Cascades, as well as in the Rocky Mountains and Coast Mountains of the United States and Canada. The diminishing frequency of Cr2 from the southern and central Sierra Nevada northward mirrors that of Cr1 in sugar pine (P. lambertiana) and points to this region as the origin of both genes. We rationalize that this coincidence may have resulted from protection that these genes may have conferred on both species to an endemic pine stem rust congeneric with C. ribicola (C. occidentale) in recent geologic epochs.     

利用UP-PCR、ISSR和AFLP标记分析玉米丝黑穗病菌遗传多样性

利用UP-PCR、ISSR和AFLP分子标记方法研究了我国主要玉米产区34株玉米丝黑穗病菌的遗传多样性。从供试引物中筛选获得具多态性的UP-PCR引物9个、ISSR引物11个和AFLP引物组合22对,分别扩增出113、72和293条谱带,多态性条带比率分别为91.15%、84.7%和83.27%。聚类分析表明,玉米丝黑穗病菌存在丰富的遗传变异,与地理来源无明显相关性。3种分子标记的遗传相似系数矩阵相关性分析表明,UP-PCR与AFLP具有较高的相关性,相关系数为0.698;UP-PCR与ISSR、ISSR与AFLP的相关系数分别为0.659和0.633。从多态性水平、稳定性和可操作性可以看出,UP-PCR技术更适于分析玉米丝黑穗病菌遗传多样性。此外,UP-PCR、ISSR和AFLP标记划分的类群与鉴别寄主划分的致病类型之间存在一定的相关性,吻合率分别为50.0%、60.0%和47.6%。     

利用AFLP分子标记和无毒基因构建小麦白粉菌遗传连锁图谱

 本研究以具有8个无毒基因差异的2个小麦白粉菌亲本菌株CPW-1和E17进行杂交,获得97个后代菌株。以此为材料,利用AFLP分子标记的方法,对小麦白粉菌亲本及其杂交后代群体进行多态性分析,应用MAPMAKER/EXP(3.0b)并结合MapDrawV2.1软件构建小麦白粉菌的遗传连锁图谱。该图谱有10个连锁群,含117个标记位点(5个无毒基因位点和112个AFLP位点),总长1425.1cM。此图谱为首个在小麦白粉菌中构建的具有117个位点的遗传连锁图谱,而且首次获得了与无毒基因紧密连锁的AFLP分子标记位点。这些工作为无毒基因的定位和克隆、为全面进行小麦白粉菌的遗传学研究奠定了基础。     

我国栽培番茄部分品种LescPtoF基因序列多态性的初步研究

根据已报道的番茄抗细菌性斑点病基因Pto家族成员之一LescPtoF的序列特征设计一对引物,通过PCR扩增,从我国10个抗病栽培番茄品种和3个感病品种获得13个同源序列。Blast结果表明,2个来自抗病品种和2个来自感病品种的核苷酸序列均和报道的LescPtoF完全一致。其余9个核苷酸序列和LescPtoF序列相似性最低为91%,它们之间相似性最低为87%。具有完整读码框的11个核苷酸序列所编码氨基酸与LescPtoF蛋白相似性最低为84%,11个氨基酸序列之间相似性最低也为84%。研究结果表明我国栽培番茄品种LescPtoF基因同源序列的分布具有多态性。     

黏虫体内两种微管蛋白基因cDNA序列的克隆与序列分析

以黏虫4龄幼虫为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),分别扩增得到该虫的α和β微管蛋白基因的cDNA序列各1条。其中α微管蛋白基因的cDNA序列1 443个碱基,包括一个1 353个碱基的开放阅读框,编码一个含450个氨基酸的蛋白,分子量约为50.0ku。氨基酸的142~148位存在一个微管蛋白标志信号片段GGGTGSG,在氨基酸序列的C-端有一个酪氨酸残基,N-端存在一个对转录后调控非常重要的保守区MRECI序列,以上特点与其他昆虫α微管蛋白氨基酸序列相同。黏虫β微管蛋白基因cDNA序列含1 906个碱基,开放读码框1 344个碱基,编码氨基酸447个,分子量约为50.2ku,等电点4.75。1~4个氨基酸MREI为β微管蛋白转录后调控信号,140~146GGGTGSG位同样存在一个微管蛋白标志信号片段。序列比对表明,克隆的α和β微管蛋白基因与其他昆虫的α和β微管蛋白基因在核苷酸和氨基酸水平上都是高度同源的,黏虫与家蚕(Bombyx mori)α微管蛋白的氨基酸序列同源性达到99.3%,与其他3种夜蛾科昆虫α微管蛋白的氨基酸序列同源性更是达到100%。黏虫与家蚕β微管蛋白的氨基酸序列同源性达到98.7%,与烟草天蛾β微管蛋白的氨基酸序列同源性达到99.6%。两个基因的cDNA序列已经登录GenBank并获得登录号分别为EU100016和EU234504。