小麦硫代硫酸硫转移酶类似基因的克隆与定位

小麦-簇毛麦6VS/6AL易位系92R137含有抗白粉病基因Pm21。为了研究该易位系的抗病机理,应用mRNA差异显示和快速扩增cDNA未端（Rapid Amplification of cDNAEnd,RACE)技术对在白粉菌诱导后表达增强的基因进行了克隆,分离到1个命名为TaTST的全长cDNA序列。Northern杂交分析表明,TaTST基因在白粉菌诱导后表达明显增强,24h达到峰值,氨基酸序列同源性分析表明,TaTST与Datisca glomerata的硫代硫酸硫转移酶基因（rho-danese,EC,2.8.1.1)序列有64%相同,80%相似,用中国春缺体/四体系和端体系Southern杂交和基因特异性引物扩增（gene specific primer-PCR)将TaTST基因定位在小麦6B染色体短臂上,Southern杂交表明,该基因为单拷贝基因,由于在杨麦5号和6VS/6AL易位系间存在明显多态,可以推测在6VS上有TaTST的同源基因,TaTST是从小麦中分离的新基因。白粉菌诱导后的表达变化提示;TaTST与小麦抗白粉病反应有关。     

普通小麦中一个类受体激酶基因的克隆、鉴定和表达分析

为研究抗白粉病小麦（Triticum aestivum L．）品系在小麦白粉病菌（Blumeria graminis f. sp．tritici）侵染后有无LRK10同源基因表达,依据小麦蛋白激酶LRK10和其它植物蛋白激酶第6亚结构域设计了一个5’-RACE兼并性引物。以接种小麦白粉病菌后的小麦抗白粉病品系“99—2439”幼苗叶片cDNA为模板进行5’-RACE扩增,获得了一个1551bp长的蛋白激酶基因cDNA片段（S1125,GenBank登录号：AY584533）。此后,通过RACE技术成功地获得了该基因的全长cDNA克隆。该克隆编码637个氨基酸组成的多肽。同源性查寻表明,该基因属于先前命名为wfrk（wheat leaf rust kinase）的小麦类受体蛋白激酶基因家族。与LRK10相似,这个新的小麦类受体蛋白激酶有5个明显的功能域：位于氨基端的疏水信号序列、推测的胞外结构域、跨膜域、高荷电序列和位于羧基端的丝氨酸／苏氨酸激酶域,因此被命名为TaLRK（Triticum aestivum LRK）。以小麦肌动蛋白基因为对照,通过半定量反转录PCR（semi—QRT—PCR）技术对叶片中TaLRK基因在小麦白粉病菌接种后的转录水平表达谱进行了研究。结果表明,小麦白粉病菌的侵染使TaLRK基因的转录显著增强。组织特异性表达分析证明,这一基因仅在小麦的绿色部分表达。研究结果提示TaLRK可能参与了小麦的抗白粉病反应。     

小麦Mlo及NBS—LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因MlocDNA序列及一个来源于栽培一粒小麦（Triticum monococcumL.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选。结果获得两个表达基因的cDNA克隆。其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%。另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类。白粉菌诱导前后,该片段RT-PCR扩增产物存在差异。表明该片段可能与小麦抗病性相关。利用“中国春”缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上。     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.     

白粉菌诱导下感病小麦京411中TCTP基因的表达分析

TCTP广泛存在于动植物及微生物中,参与重要的生物过程。对小麦中克隆到的TCTP基因进行了白粉菌诱导下的表达模式分析,结果发现,在对白粉菌敏感的小麦京411中,白粉菌侵染后TCTP基因应答迅速,且基因表达水平较高。用病毒介导的基因沉默方法沉默京411中的TCTP基因,发现白粉菌成功侵染小麦叶片的比例下降,乳突等抗性结构特征比例上升,H2O2在细胞内的累积水平升高。试验结果表明,TCTP在小麦与白粉菌互作过程中起重要作用。     

小麦抗白粉病相关基因的转化

利用玉米花青素苷合成调节基因C1-Lc作为报告基因, 通过瞬间表达后愈伤组织表面红色斑点的统计分析, 优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是2个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的2个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中, 使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株, 进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株, 转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明, 外源基因的导入不同程度上增强了植株的白粉病抗性, 表现为延缓了白粉菌的发育。利用玉米花青素苷合成调节基因C1-Lc作为报告基因,通过瞬间表达后愈伤组织表面红色斑点的统计分析,优化了小麦幼胚愈伤组织的基因枪转化参数。小麦Beclin1类似基因TaTBL和硫代硫酸硫转移酶基因TaTST是两个在白粉菌诱导条件下具有增强表达特性的抗病相关基因。本实验进一步利用基因枪将ubi强启动子控制下的两个基因导入到小麦品种扬麦158的幼胚愈伤组织细胞中,使用除草剂经两轮选择培养基上的筛选和再生获得抗性植株,进一步通过抗性植株的PCR分析获得转TaTBL基因植株5株,转TaTST基因植株6株。转基因植株离体叶片的人工接种实验表明,外源基因的导入不同程度上增强了植株的白粉病抗性,表现为延缓了白粉菌的发育。     

小麦锌指蛋白基因的克隆、序列与表达分析

根据R基因及其调控基因保守序列设计简并性引物,对白粉菌接种和未接种处理的一对抗病和感病的小麦—黑麦等位突变易位系TAM104R和TAM104S总RNA进行RT-PCR扩增,得到一个诱导表达的cDNA片段。序列分析表明,该片段全长2474bp,其中含有一个822bp的完整开放阅读框,推测其编码一个有273个氨基酸残基、分子量约31kD的蛋白质分子。蛋白质的氨基酸序列比对显示,该蛋白质分子具有C2HC锌结合motif CX2CX4HX4C结构和锌指domain,可见克隆的cDNA是一个锌指蛋白基因,命名为TaZF。Southern杂交表明,TaZF在抗病易位系TAM104R的基因组中是多拷贝的。半定量RT-PCR分析显示,TaZF基因属组成型表达、但受白粉菌诱导表达上调的基因,推测其与白粉病菌的侵染过程相关。基因组DNA专化扩增、克隆和测序揭示TaZF基因无内含子。     

谷胱甘肽硫转移酶基因表达的调控

催化内源性或外源性亲电子化合物与谷胱甘肽(GSH)结合的谷胱甘肽硫转移酶(GST)超基因家族是一族解毒功能蛋白.其基因的表达通过不同的机制受多种物质的调控.根据最近文献资料,对调控谷胱甘肽硫转移酶基因表达的基因结构、调控机制及氧化应激对谷胱甘肽硫转移酶基因表达的调控作用等作一简要综述.     

葡萄风信子谷胱甘肽S转移酶基因克隆与表达分析

以亚美尼亚葡萄风信子为材料,利用本课题组前期获得的葡萄风信子转录组数据库,根据已经获得的葡萄风信子GST基因片段,通过PCR技术克隆得到葡萄风信子GST基因的c DNA序列,命名为Ma GST。Ma GST的c DNA全长为711 bp,开放阅读框为666 bp,编码221个氨基酸,推测蛋白质分子量为54.1 k D,理论等电点p I为5.13。利用生物信息分析软件对Ma GST基因进行同源性比对和系统进化分析表明,结果显示该基因编码的氨基酸具有谷胱甘肽S转移酶典型的C端与N端双结构域,属于GST Tau家族蛋白;与洋葱和小麦GST基因的一致性分别为76.72%和62.50%。实时定量PCR结果显示Ma GST在葡萄风信子各组织器官表达强度相似,属于组成型表达;水杨酸(SA)可以明显诱导Ma GST表达,Ma GST对氯化钠(Na Cl)应答不是很明显,甚至表达量有稍微的下调。     

利用瞬间表达技术分析小麦抗病相关基因的功能

采用瞬间表达技术分析了TaTBL、TaPK1和TaTST等3个抗病相关基因的功能。首先将这3个小麦抗病相关基因构建入高效表达载体,然后使用基因枪将目标基因和GUS基因载体同时导入到感白粉病小麦品种离体叶片表皮细胞中,用GUS基因标记阳性转化细胞。转化后接种白粉菌孢子,48h后观察转化阳性表皮细胞,研究抗病相关基因表达对白粉菌入侵及吸器形成产生的影响。结果表明,这3个基因在感病小麦品种叶片表皮细胞中的瞬间表达,对白粉菌侵入和吸器形成均有部分抑制作用,在一定程度上增强了表达细胞对白粉菌的抗性。     

基于SSU序列的串珠藻目植物系统发育分析

通过18S rDNA基因(SSU)序列,构建了串珠藻目植物的系统发育关系.结果显示:SSU基因序列片段长度为1 871 bp,核苷酸变异位点有709个,占序列长度的38％;其中简约信息位点有169个,占序列长度的9％.用最大似然法、邻接法和贝叶斯法构建的系统树拓扑结构基本一致,都显示红索藻目的2个属独立于串珠藻目成单独分支,支持红索藻目的建立;胶串珠藻独立于其他串珠藻组植物,支持将其单独分组;数据同时支持将扭曲组和杂生组合并,建立Kumanoa属;但多芒组、绿色组、沼生组等因分子序列数据涉及的种类较少,其系统关系的确定还需要更多的证据.     

串珠藻目植物的系统发育-基于rbcL序列的证据

世界范围内报道的全部串珠藻目种类均生活于淡水中,而在淡水红藻中,70%约有130种属于串珠藻目。研究以目前获取的来自世界各大洲串珠藻目植物43种的rbcL基因序列,结合其形态和生物地理特征,构建了该目的系统发育关系,以期探讨整个串珠藻目植物的系统发育关系及发生途径,进而为研究该目以至淡水红藻的起源提供基本资料。运用PAUP*4.0b10和MrBayes 3.0b4等软件对43种串珠藻目植物的叶绿体DNA rbcL基因序列进行系统发育分析,探讨了其主要分类群的系统演化关系。用最大简约法、邻接法和贝叶斯分析方法构建的系统树基本一致,结果显示:(1)基于分子数据分析结果显示,红索藻目植物均独立于串珠藻目植物,构成一个单独的分支,支持红索藻目的建立。(2)鱼子菜科属于串珠藻目植物中较为进化的类群。(3)串珠藻属扭曲组与杂生组的差异度较小,结合其形态特点,倾向于将杂生组并入扭曲组。(4)串珠藻科属于串珠藻目中最大的科,包括较多的种类,其系统关系也较为复杂。因此,串珠藻科系统发育关系的明确有待于进一步结合更多的分子数据和形态学特征加以分析研究。       

基于叶绿体psaA和psbA基因的中国熊野藻属植物系统发育分析

以中国产熊野藻属Kumanoa的两个种, 绞扭熊野藻K. intorta (=绞扭串珠藻Batrachospermum intortum), 弯形熊野藻K. curvata (=弯形串珠藻B. curvatum)和其他6种淡水红藻为实验材料, 对其psaA和psbA基因进行扩增和测序, 并与GenBank中相近序列进行比对分析, 以贝叶斯法、最大似然法和邻接法分别构建了单基因和联合基因系统发育树. 结果表明, 3种方法构建的系统树具有相似的拓扑结构, 反映的系统发育关系基本一致, 熊野藻属中的两个种聚为一支, 与串珠藻属相分离, 支持该属的建立; 中国产的熊野藻属分子学研究结果与来自南美洲及澳洲的该属植物结果一致, 说明该属的建立具有广泛的地理适用性. 系统发育树聚类结果也明确反映了熊野藻属与串珠藻属较近的亲缘关系, 根据果胞枝形态特点, 推测熊野藻属进化地位晚于串珠藻属植物, 而早于顶丝藻目和红索藻目. 此外, 胶串珠藻与其他串珠藻组植物分离, 支持将其单独分组, 红索藻目植物与串珠藻目植物分离, 支持红索藻目的建立. 同时也表明psaA和psbA基因用于淡水红藻分析, 能够较好地反映其系统发育关系.       

PHYLOGENETIC AFFINITIES OF “CHANTRANSIA” STAGES IN MEMBERS OF THE BATRACHOSPERMALES AND THOREALES (RHODOPHYTA)1

This study evaluated the phylogenetic relationship among samples of “Chantransia” stage of the Batrachospermales and Thoreales from several regions of the world based on sequences of two genes—the plastid‐encoded RUBISCO LSU gene (rbcL) and the nuclear SSU ribosomal DNA gene (SSU rDNA). All sequences of “Chantransia macrospora” were shown to belong to Batrachospermum macrosporum based on both molecular markers, confirming evidence from previous studies. In contrast, nine species are now associated with “Chantransia pygmaea,” including seven species of the Batrachospermales and two of the Thoreales. Therefore, the presence of “C. macrospora” in a stream can be considered reliable evidence that it belongs to B. macrosporum, whereas the occurrence of “C. pygmaea” does not allow the recognition of any particular species, since it is associated with at least nine species. Affinities of “Chantransia” stages to particular taxa were congruent for 70.5% of the samples comparing the rbcL and SSU analyses, which were associated with the same or closely related species for both markers. Sequence divergences have been reported in the “Chantransia” stage in comparison to the respective gametophyte, and this matter deserves further attention.     

A PROPOSAL FOR A NEW RED ALGAL ORDER,THE THOREALES1

Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well‐supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate‐like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate‐like.     

Investigation of a freshwater acrochaetioid alga (Rhodophyta) with molecular and morphological methods

An enigmatic acrochaetioid alga was collected from Niangziguan spring in Shanxi Province, northern China. Morphological data indicated that this alga reproduces exclusively asexually by monosporangia and its morphological characteristics suggested that it might be referred to Audouinella heterospora. To ascertain its phylogenetic position, phylogenetic trees were reconstructed using partial sequences of the plastid‐encoded gene (rbcL) and the nuclear‐encoded gene (SSU rDNA) applying Bayesian inference (BI), maximum parsimony (MP) and maximum likelihood (ML). However, phylogenetic reconstructions showed that this acrochaetioid alga does not belong in a clade with the genus Audouinella, but forms a clade with Thorea hispida (Thoreales). Based on this analysis it is concluded that A. heterospora represents the ‘chantransia’ stage of T. hispida.     

A Three-Gene Dinoflagellate Phylogeny Suggests Monophyly of Prorocentrales and a Basal Position for <Emphasis Type="Italic">Amphidinium</Emphasis> and <Emphasis Type="Italic">Heterocapsa</Emphasis>

Many outstanding questions about dinoflagellate evolution can potentially be resolved by establishing a robust phylogeny. To do this, we generated a data set of mitochondrial cytochrome b (cob) and mitochondrial cytochrome c oxidase 1 (cox1) from a broad range of dinoflagellates. Maximum likelihood, maximum parsimony, and Bayesian methods were used to infer phylogenies from these genes separately and as a concatenated alignment with and without small subunit (SSU) rDNA sequences. These trees were largely congruent in topology with previously published phylogenies but revealed several unexpected results. Prorocentrum benthic and planktonic species previously placed in different clusters formed a monophyletic group in all trees, suggesting that the Prorocentrales is a monophyletic group. More strikingly, our analyses placed Amphidinium and Heterocapsa as early splits among dinoflagellates that diverged after the emergence of O. marina. This affiliation received strong bootstrap support, but these lineages exhibited relatively long branches. The approximately unbiased (AU-) test was used to assess this result using a three-gene (cob + cox1 + SSU rDNA) DNA data set and the inferred tree. This analysis showed that forcing Amphidinium or Heterocapsa to relatively more derived positions in the phylogeny resulted in significantly lower likelihood scores, consistent with the phylogenies. The position of these lineages needs to be further verified. Reviewing Editor: Dr. Martin Kreitman     

采自吉林省敦化市的2个蘑菇目(Agaricales)新记录种

【背景】在中国吉林省延边朝鲜族自治州敦化市寒葱岭红叶谷进行大型真菌资源调查的过程中发现了2个物种。【目的】确定这2个物种的分类地位。【方法】采集大型真菌标本,对其形态进行详细观察和描述,并提取DNA、测定rDNA ITS序列,基于最大似然法和贝叶斯法构建分子系统发育树。【结果】鉴定结果表明,这2个蘑菇目物种分别是径边刺毛鬼伞(Tulosesus callinus)和拉氏红菇(Russula lakhanpalii),在形态上与原始描述高度吻合,系统发育分析也验证了形态学的鉴定结果。【结论】拉氏红菇(R. lakhanpalii)系中国首次报道,径边刺毛鬼伞(T. callinus)为吉林省新记录种。     

Phylogeny of the East Asian botiine loaches (Cypriniformes,Botiidae) inferred from mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> gene sequences

The Botiinae have traditionally represented a subfamily of the Cobitidae. At present, the classification and phylogenetic relationships of the Botiinae are controversial. To address systematic and phylogenetic questions concerning this group, we sequenced the complete cytochrome b gene from 34 samples, of which 24 represented 13 species of the East Asian botiine fishes, while the other 10 were non-botiine loach species. For the 1140 bp sequences determined, 494 sites were variable ones, of which 424 were parsimony informative. With Myxocyprinus asiaticus as an outgroup, molecular phylogenetic trees were constructed using the neighbor-joining, maximum parsimony, maximum likelihood and Bayesian methods. All molecular phylogenetic trees revealed that botiine fishes form a monophyletic group and are distantly related to other loaches, suggesting that the Botiinae should be placed in their own family. Within the Botiinae, there are three genera; Botia, Parabotia, andLeptobotia, each genus forming a monophyletic group, with the genus Botia as the most ancestral split. Our molecular results are in agreement with morphological analyses of botiines, suggesting that Botia is the ancestral genus, while Leptobotia and Parabotia were resolved as more derived sister groups.