番茄RGA4蛋白与马铃薯Rpi-blb1蛋白的同源比较分析

为获得番茄抗晚疫病广谱性基因信息,采用同源电子克隆法,基于番茄蛋白序列数据库,以马铃薯晚疫病广谱抗性蛋白Rpi-blb1为种子序列,获得番茄疾病抗性蛋白RGA4,并进行基因电子定位和基因结构、模体、二级结构、基因进化及基因电子表达等分析,以证明两者的进化关系。结果表明:番茄RGA4蛋白(XP_004245923.1)序列具有NB-ARC和LRR8两个保守结构域,定位于第8条染色体SL2.40区间,相应基因位于8号染色体序列的57228847-57232935 bp区间,长度为4 089 bp,由2个外显子和1个内含子组成,编码988个氨基酸序列,该蛋白为不稳定分泌球形蛋白;番茄RGA4蛋白和马铃薯Rpi-blb1蛋白在二级结构分布、基因序列组成、基因定位等方面相似性较高,可确定两者为垂直同源关系,但在基因进化和模体组成方面存在差异,可能导致两者功能上的不同。研究结果可为番茄晚疫病广谱抗性基因克隆及其利用提供理论依据。     

番茄中Rpi-blb2同源基因的挖掘与鉴定

番茄晚疫病是番茄生产中的主要病害之一,经常会造成较大的经济损失。晚疫病生理小种的变异和进化常会导致番茄品种原有的遗传抗性丧失,因此不断挖掘新的抗性基因,改良番茄晚疫病抗性是番茄抗病育种的长期任务。该研究采用BLAST同源比对的方法,以马铃薯野生近缘种的晚疫病抗性蛋白序列Rpi-blb2为种子序列,在NCBI蛋白质序列数据库中检索得到11条番茄蛋白质序列,这些序列与种子序列相似性为78%~83%,属于番茄疾病抗性蛋白家族,并对该家族成员进行了基因结构、基因定位、序列保守结构域和进化关系等分析。结果表明:该家族中10条序列分布在第Ⅵ条染色体上,1条分布在第Ⅴ染色体上;6号染色体上的10序列呈现2个抗病基因簇分布,在染色体上分别占据2个和3个基因位点;10条同源蛋白是Rpi-blb2的共同垂直同源蛋白,但不具有平行同源关系,大多数成员定位于细胞质。按照蛋白质保守结构域和基因定位的不同可分为三类,第一类共4条系列,包含有DUF3542和NB-ARC两个保守结构域特征序列;第二类共6条序列,与马铃薯Rpi-blb2蛋白一样,仅包含NB-ARC保守结构域特征序列,在这2类蛋白序列的NB-ARC结构域均位于序列中部;第三类(仅包含XP_004239406.1)虽然也具有与第一类蛋白相似的DUF3542和NB-ARC结构域,但在结构域两端的非保守区序列较短,且位于5号染色体上,因此将其单独归为1类。前两类蛋白成员相应的基因具有1~2个内含子,第3类蛋白不含内含子。该研究结果为利用生物技术选育番茄抗性品种提供了理论基础。     

马铃薯种质资源的晚疫病抗性鉴定

采用离体叶片接种法,对43份马铃薯种质资源进行晚疫病抗性鉴定、比较和评价。以接种5 d后的感病品种‘Désirée’和抗病品种‘加湘1号’叶片症状为对照,鉴定出5份表现为高抗的种质资源,其中包含3个Solanum phurejia和2个S. tuberosum ssp. andigena材料。另外,还鉴定出14份中抗材料(No. 7～20)。结果表明,野生种和安第斯山栽培亚种马铃薯资源的抗性材料较为丰富,可作为晚疫病抗病育种的亲本。     

马铃薯晚疫病抗病基因研究进展

马铃薯晚疫病是马铃薯和番茄等茄科植物中最主要的病害之一,每年都引起巨大的经济损失。基因工程技术的发展为马铃薯晚疫病的防治工作提出了新的契机,从晚疫病抗性品种中筛选出具有高持久抗性的抗病基因,并将其转化到栽培品种中去,无疑是我们开发持久性晚疫病抗性的最快捷的手段。到目前为至,已经有十几个晚疫病抗性基因从S．demissum,S. bulbocastanum,S．berthauhii,S．mochiquense和S．pinnatisectum等抗性马铃薯品种中鉴定出来并已定位在马铃薯染色体基因组上,并有4个被克隆出来（R1,R3a,Rpi—blb1／RB和Rpi—blb2）。主要概述了马铃薯晚疫病抗病基因的研究现状和发展前景。     

马铃薯晚疫病抗性基因分子标记检测及抗性评价

马铃薯是重庆优势特色作物之一,但其安全生产受到晚疫病的严重威胁。马铃薯生产中种植抗病品种是防控马铃薯晚疫病最为经济有效和环境友好的途径。为了了解来自国内外不同种质的晚疫病抗性基因组成以及确定在重庆市具有晚疫病抗性的马铃薯品种(系),本研究以218份来自国内外的品种(系)为材料,进行了6个晚疫病抗性(R)基因分子标记检测,同时进行了田间晚疫病抗性评价及室内接种鉴定和筛选。研究结果显示,6个R基因的分子标记在供试材料中均有分布,但分子标记的组成不尽相同,主要分为4大类。第Ⅰ类含有具有广谱抗性基因的RB,晚疫病抗性评价表现为中抗以上;第Ⅲ类缺失R2 family基因标记,绝大部分表现为晚疫病敏感型;第Ⅱ类和第Ⅳ类中分别主要含有3个R基因(R2 Family+R3a+R3b)标记类型和4个R基因(R1+R2 Family+R3a+R3b)标记类型,这两类材料中表现出一定比例的晚疫病抗性水平,但第Ⅳ类中出现抗性表现的比例高于第Ⅱ类。结果说明含有RB基因标记贡献了较高的晚疫病抗性,缺失R2 famlily基因标记的材料可能不利于晚疫病抗性,利用这些基因标记辅助筛选有助于提高重庆地区晚疫病抗性育种效率。本研究评价了218份马铃薯材料6个重要R基因组成,并筛选出重庆地区表现抗性的多个材料,为新品种(系)的推广应用以及抗病育种选育提供了科学依据,同时为发掘新的抗病基因提供了遗传资源。     

哺乳动物cd59基因的进化

采用PCR以及BLAST方法从5种哺乳动物中获得了cd59基因的编码区序列, 结合 GenBank中已有的序列, 计算cd59基因在哺乳动物中的核苷酸替换速率. 对非同义替换速率和同义替换速率进行比较的结果显示, cd59在哺乳动物中总体上受到负选择作用; 用PAML软件“位点-特异”模型检测到4个受到正选择作用的位点, 4个位点分布于分子表面, 其中2个位于功能重要的区域; 此外, 用“支-位点-特异”模型在小鼠通过基因复制后形成的cd59a和cd59b上检测正选择引起的加速进化, 并检测到该支系特异的正选择位点1个.     

四川省马铃薯晚疫病菌群体表型和遗传变异的分析

致病疫霉Phytophthora infestans引起的晚疫病是马铃薯的一种毁灭性病害。为了对四川省近年马铃薯晚疫病菌进行系统多样性分析,本研究从表现型和基因型两个方面鉴定了四川省马铃薯晚疫病菌的群体多态性。交配型、甲霜灵敏感性和生理小种的鉴定结果发现A1交配型菌株只有2份,A2型有29份,自育型12份;甲霜灵敏感菌株3株,中抗菌株22株,高抗菌株18株;共测定出11个生理小种,其中全毒力小种发生频率最高,占供试菌株的25.58%。基因型鉴定结果表明,43份材料共发现了10种SSR基因型,5个SSR标记在该种群上共产生了16个SSR位点,每个标记平均有3.2个位点,多态性信息含量平均值为0.46,SSR4是多态性最丰富的标记。本试验中A2菌株和自育型的基因型SSR相同,其中全毒力小种是当地的优势致病菌,该研究为今后四川马铃薯晚疫病的有效防控提供理论依据。     

甘薯栽培种及其近缘野生种的DAPI核型及rDNA FISH分析

利用DAPI显带和rDNA-FISH技术对栽培种甘薯(‘徐薯18’)(Ipomoea batatas cv.Xushu No.18)及2种不同产地近缘野生种(Ipomoea hederacea Jacq.)进行了细胞遗传学研究。DAPI核型分析表明,‘徐薯18’核型公式为2n=6x=90=72m+18sm(18SAT),随体位于第1、3、6染色体上;美国近缘野生种核型公式为2n=2x=30=30m(4SAT),香港近缘野生种核型公式为2n=2x=30=20m+10sm(4SAT),随体均位于第6、12染色体上。rD-NA-FISH结果显示,栽培种甘薯基因组中含有3对5SrDNA位点,分别位于着丝粒区、亚着丝粒区和染色体端部;美国近缘野生种基因组中含有2对5SrDNA位点,香港近缘野生种基因组中含有1对5SrDNA位点,均位于随体部位;两种不同地域来源的近缘野生种基因组中均含有2对45SrDNA位点,分别位于第6和第12染色体上。     

马铃薯泛素结合酶基因StUBC17的克隆与功能分析

泛素结合酶(Ubiquitin-conjugating Enzyme E2,UBC)与植物生长发育和抗病反应密切相关。为了解泛素结合酶基因对植物抗晚疫病的贡献,从马铃薯栽培种"合作88"中克隆出一个E2泛素结合酶基因,命名为StUBC17,并对其受晚疫病菌诱导表达特性及功能进行分析。利用病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)技术降低本氏烟(Nicotiana benthamiana)中StUBC17同源基因(NbUBC)的转录水平,再接种晚疫病菌进行抗病性鉴定。进一步在基因沉默植株上瞬时表达马铃薯抗病基因和其相应的无毒基因(R3a+AVR3a、R3b+AVR3b和Rx+CP)及INF1。StUBC17编码区全长447 bp,编码148个氨基酸,其蛋白分子量为16.52 kD,理论等电点为7.72。表达分析结果表明,StUBC17受晚疫病菌诱导表达。抗病鉴定结果显示,与对照植株相比,NbUBC沉默植株的抗病性显著降低。沉默NbUBC不影响过敏反应(Hypersensitive responses,HR)的发生。StUBC17是植物防御晚疫病所需,但该基因的沉默并不影响R3a、R3b、Rx及INF1介导的HR反应。     

二倍体蔷薇FT基因序列多样性研究

以3个类群73个二倍体蔷薇属(Rosa)植物为材料,克隆获得其FLOWERING LOCUS T(FT)同源基因,并对该基因的编码区序列进行多态性分析以及多维尺度(MDS)聚类分析。结果显示,73个二倍体蔷薇植物的FT基因共检测到215个核苷酸多态性位点,其中包括214个SNP和1个缺失突变,平均185个碱基发生1次突变;氨基酸多态性分析结果显示共有35个氨基酸发生变异,平均379.6个氨基酸残基发生1次突变;突变位点统计分析结果发现39、258、426 bp位点是高频突变位点,其碱基由A或C突变为T。MDS聚类分析结果表明,3个类群FT基因编码区序列的碱基组内差异依次排序为:野生种月季组中国古老月季,氨基酸组内差异依次排序为:中国古老月季月季组野生种,推测中国古老月季在长期栽培驯化过程中,其FT基因可能经历了较强的人工选择压力,月季组的种和变种可能是古老月季的重要亲本来源。     

Nucleotide polymorphism and molecular evolution of the LRR region in potato late blight resistance gene Rpi-blb2

Rpi-blb2, which is originally derived from Solanum bulbocastanum, is a broad-spectrum potato late blight resistance gene and belongs to the NBS-LRR family. Here, the LRR homologues of Rpi-blb2 were cloned with PCR method from 40 potato cultivars (including 20 resistant potato cultivars and 20 susceptible ones) and 7 wild potato populations. Then, the similarities of the sequences, polymorphic (segregating) sites, and nucleotide diversities were estimated by bioinformatic methods. The results showed that high nucleotide polymorphism and some hot-spot mutations existed in the LRR region of Rpi-blb2. The test of Ka/Ks ratio showed that the function of LRR was conserved because of the purifying selection, although different positions of the Rpi-blb2 LRR region were under different selection pressures. Moreover, the LRR region of Rpi-blb2 had no clear differentiation between the cultivated and wild potatoes.     

An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease for potato cultivation. Here, we describe the positional cloning of the Rpi-blb1 gene from the wild potato species Solanum bulbocastanum known for its high levels of resistance to late blight. The Rpi-blb1 locus, which confers full resistance to complex isolates of P. infestans and for which race specificity has not yet been demonstrated, was mapped in an intraspecific S. bulbocastanum population on chromosome 8, 0.3 cM from marker CT88. Molecular analysis of a bacterial artificial chromosome (BAC) clone spanning the Rpi-blb1 locus identified a cluster of four candidate resistance gene analogues of the coiled coil, nucleotide-binding site, leucine-rich repeat (CC-NBS-LRR) class of plant resistance (R) genes. One of these candidate genes, designated the Rpi-blb1 gene, was able to complement the susceptible phenotype in a S. tuberosum and tomato background, demonstrating the potential of interspecific transfer of broad-spectrum late blight resistance to cultivated Solanaceae from sexually incompatible host species. Paired comparisons of synonymous and non-synonymous nucleotide substitutions between different regions of Rpi-blb1 paralogues revealed high levels of synonymous divergence, also in the LRR region. Although amino acid diversity between Rpi-blb1 homologues is centred on the putative solvent exposed residues of the LRRs, the majority of nucleotide differences in this region have not resulted in an amino acid change, suggesting conservation of function. These data suggest that Rpi-blb1 is relatively old and may be subject to balancing selection.     

The late blight resistance locus Rpi-bib3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpi-abpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.     

The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato

The necessity to develop potato and tomato crops that possess durable resistance against the oomycete pathogen Phytophthora infestans is increasing as more virulent, crop-specialized and pesticide resistant strains of the pathogen are rapidly emerging. Here, we describe the positional cloning of the Solanum bulbocastanum-derived Rpi-blb2 gene, which even when present in a potato background confers broad-spectrum late blight resistance. The Rpi-blb2 locus was initially mapped in several tetraploid backcross populations, derived from highly resistant complex interspecific hybrids designated ABPT (an acronym of the four Solanum species involved:S. acaule, S. bulbocastanum, S. phureja and S. tuberosum), to the same region on chromosome 6 as the Mi-1 gene from tomato, which confers resistance to nematodes, aphids and white flies. Due to suppression of recombination in the tetraploid material, fine mapping was carried out in a diploid intraspecific S. bulbocastanum F1 population. Bacterial artificial chromosome (BAC) libraries, generated from a diploid ABPT-derived clone and from the resistant S. bulbocastanum parent clone, were screened with markers linked to resistance in order to generate a physical map of the Rpi-blb2 locus. Molecular analyses of both ABPT- and S. bulbocastanum-derived BAC clones spanning the Rpi-blb2 locus showed it to harbor at least 15 Mi-1 gene homologs (MiGHs). Of these, five were genetically determined to be candidates for Rpi-blb2. Complementation analyses showed that one ABPT- and one S. bulbocastanum-derived MiGH were able to complement the susceptible phenotype in both S. tuberosum and tomato. Sequence analyses of both genes showed them to be identical. The Rpi-blb2 protein shares 82% sequence identity to the Mi-1 protein. Significant expansion of the Rpi-blb2 locus compared to the Mi-1 locus indicates that intrachromosomal recombination or unequal crossing over has played an important role in the evolution of the Rpi-blb2 locus. The contrasting evolutionary dynamics of the Rpi-blb2/Mi-1 loci in the two related genomes may reflect the opposite evolutionary potentials of the interacting pathogens.     

Genetic diversity among late blight resistant and susceptible potato genotypes

RAPD polymerase chain reaction analysis was used to study the genetic diversity among a wild potato variety Solanum demissum (very resistant to late blight) and six potato cultivars (Hanna, Lady-Olympia, Lady-Rosetta, Spunta, Diamant and Cara) varied in their resistance to Phytophthora infestans. Cluster analysis of six potato genotypes showed that, all tested genotypes were separated into two clusters (1 and 2). Cluster 1, included only the wild potato variety (S. demissum), whereas cluster 2 divided into two groups (G1 and G2). Late blight high resistant cultivars Hanna and Cara were grouped in G1. Group 2 included the moderate resistant cultivar Spunta and the susceptible cultivars Diamant, Lady-Rosetta and Lady-Olympia. The potato cultivars that showed highest genetic similarity to the wild potato variety were the resistant cultivars Hanna and Cara. Lowest genetic similarity was obtained with the susceptible cultivars Lady-Rosetta, Diamant and Lady-Olympia. RAPD primer K17 yielded a band with molecular weight of 936 bp found in all susceptible potato cultivars (Lady-Rosetta, Lady-Olympia and Diamant). On the other hand, band with molecular weight of 765 bp were detected in the wild potato and the resistant cultivars Hanna and Cara. Results of this study suggested that, the RAPD marker technique could be beneficial for revealing the genetic variability of different genotypes of potato varied in their resistibility to late blight.     

Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties.     

甘薯属植物过氧化物酶同工酶分析

采用垂直平板聚丙烯酰胺凝胶电泳技术,对23份甘薯属不同倍性材料进行过氧化物酶同工酶酶谱分析。初步结果表明,过氧化物酶同工酶酶带数目与材料倍性无明显相关性;二倍体或四倍体野生种的种间酶谱差异显著;六倍体野生种不同株系间以及六倍体栽培种甘薯的不同品种间酶谱差异较小;但栽培种甘薯与六倍体野生种I.trifida(6x)、四倍体野生种I.littoralis(4x)以及二倍体野生种I.trifida(2x)的酶谱有4条明显共同标记带,表明其间有一定亲缘关系。     

Dramatic Number Variation of R Genes in Solanaceae Species Accounted for by a Few R Gene Subfamilies

Most disease resistance genes encode nucleotide-binding-site (NBS) and leucine-rich-repeat (LRR) domains, and the NBS-LRR encoding genes are often referred to as R genes. Using newly developed approach, 478, 485, 1,194, 1,665, 2,042 and 374 R genes were identified from the genomes of tomato Heinz1706, wild tomato LA716, potato DM1-3, pepper Zunla-1 and wild pepper Chiltepin and tobacco TN90, respectively. The majority of R genes from Solanaceae were grouped into 87 subfamilies, including 16 TIR-NBS-LRR (TNL) and 71 non-TNL subfamilies. Each subfamily was annotated manually, including identification of intron/exon structure and intron phase. Interestingly, TNL subfamilies have similar intron phase patterns, while the non-TNL subfamilies have diverse intron phase due to frequent gain of introns. Prevalent presence/absence polymorphic R gene loci were found among Solanaceae species, and an integrated map with 427 R loci was constructed. The pepper genome (2,042 in Chiltepin) has at least four times of R genes as in tomato (478 in Heinz1706). The high number of R genes in pepper genome is due to the amplification of R genes in a few subfamilies, such as the Rpi-blb2 and BS2 subfamilies. The mechanism underlying the variation of R gene number among different plant genomes is discussed.     

Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode Globodera rostochiensis, based on a candidate gene approach

The endoparasitic root cyst nematode Globodera rostochiensis causes considerable damage in potato cultivation. In the past, major genes for nematode resistance have been introgressed from related potato species into cultivars. Elucidating the molecular basis of resistance will contribute to the understanding of nematode-plant interactions and assist in breeding nematode-resistant cultivars. The Gro1 resistance locus to G. rostochiensis on potato chromosome VII co-localized with a resistance-gene-like (RGL) DNA marker. This marker was used to isolate from genomic libraries 15 members of a closely related candidate gene family. Analysis of inheritance, linkage mapping, and sequencing reduced the number of candidate genes to three. Complementation analysis by stable potato transformation showed that the gene Gro1-4 conferred resistance to G. rostochiensis pathotype Ro1. Gro1-4 encodes a protein of 1136 amino acids that contains Toll-interleukin 1 receptor (TIR), nucleotide-binding (NB), leucine-rich repeat (LRR) homology domains and a C-terminal domain with unknown function. The deduced Gro1-4 protein differed by 29 amino acid changes from susceptible members of the Gro1 gene family. Sequence characterization of 13 members of the Gro1 gene family revealed putative regulatory elements and a variable microsatellite in the promoter region, insertion of a retrotransposon-like element in the first intron, and a stop codon in the NB coding region of some genes. Sequence analysis of RT-PCR products showed that Gro1-4 is expressed, among other members of the family including putative pseudogenes, in non-infected roots of nematode-resistant plants. RT-PCR also demonstrated that members of the Gro1 gene family are expressed in most potato tissues.