路易斯碱催化还原胺化反应研究

胺及其衍生物是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体。 直接还原胺化由于其合成步骤简单而成为制备二级胺和三级胺的简便方法。为了发展一种较为简便的直接还原胺化反应，我们把研究的重点放在开发一种简便实用的有机小分子催化方法上。由文献调研可知，现已报道的直接还原胺化方法大多是催化醛或酮与一级胺或者脂肪二级胺的直接还原胺化，而醛或酮与芳香二级胺的直接还原胺化却尚无报道。在本文中，我们发现用简单的四甲基乙二胺(TEMED)在室温下以二氯甲烷为溶剂即可催化三氯氢硅对酮和芳香二级胺之间的直接还原胺化反应，并取得了高达92%的收率。该反应条件温和，底物普适性广，各种类型的酮均可以与芳香二级胺进行直接还原胺化，并且得到比较满意的收率。 同时，我们从手性Sulfoximine出发，设计和合成了一系列的Sulfoximine类新衍生物，并将其应用于间接还原胺化反应中。遗憾的是我们并没有得到预期的不对称催化效果。 Amines and their derivatives are basic structural motifs in natural products and pharmaceuticals and highly versatile building blocks for various organic substrates. Direct reductive amination (DRA) is a convenient method for the preparation of secondary and tertiary amines owing to its operational simplicity. In an effort to develop a simple and convenient procedure for direct reductive amination reaction, we focused our study on search for a mild and efficient organocatalytic system. In the literature, there are many reports concerning DRA between aldehydes or ketones and either primary amines or secondary aliphatic amines. But there are no reports concerning DRA between aldehydes or ketones and secondary aromatic amines. In this study, we have developed a highly practical method for the synthesis of tertiary amines by the direct reductive amination of ketones and secondary aromatic amines with tetramethylethylenediamine (TEMED) as the catalyst using HSiCl3 as the reducing agent in dichloromethane (affording up to 92% yield). This method can be carried out under mild conditions and is compatible with many functional groups. A variety of ketones were efficiently aminated with secondary aromatic amines to afford the corresponding amines in good to excellent yields. Starting from chiral sulfoximine, we designed and synthesized a series of new sulfoximine derivatives and tested their efficiencies as asymmetric organocatalysts for the reduction of imines, which, unfortunately, only exhibited low catalytic activity and enantioselectivity.

黄龙世界自然遗产地岷江冷杉原始林群落结构与植物多样性：生境差异性研究

黄龙世界自然遗产地岷江冷杉林(Abies faxoniana)生境类型多样，群落结构复杂，群落植物种类组成多样性丰富。揭示不同生境的生物多样性及其差异是认识生物多样性格局、形成及维持机制的前提和进行多样性保育的基础。本文采用样方法对黄龙钙化滩生境、阴坡非钙化生境及半阳坡非钙化生境的岷江冷杉原始林植物群落结构及植物多样性进行了研究。结果表明： 黄龙岷江冷杉林具有明显的复层异龄结构，垂直结构明显，乔木、灌木、草本、苔藓层次分明。共发现高等植物386 种，其中维管植物46 科103 属163 种，苔藓植38 科83 属物223 种。各层片结构及物种组成如下: (1)钙化滩生境、阴坡非钙化生境、半阳坡非钙化生境分别发现乔木18 种、13种、8 种。乔木层均可分为两个亚层，第一亚层优势种均为岷江冷杉，第二亚层主要为岷江冷杉异龄树或其它大高位芽物种。钙化滩生境第一亚层除优势种岷江冷杉外混生有巴山冷杉(Abies fargesii)、粗枝云杉(Picea asperata)以及阔叶树种白桦(Betula platyphylla)等，第二亚层主要为岷江冷杉异龄树；阴坡非钙化生境第一亚层除优势种岷江冷杉外间有巴山冷杉和白桦，第二亚层物种主要为川滇长尾槭(Acer caudatum var. prattii)；半阳坡非钙化生境第一亚层除优势种岷江冷杉外混生有巴山冷杉，第二亚层主要为岷江冷杉异龄树。依乔木层优势种的差异，钙化滩生境及半阳坡非钙化生境为岷江冷杉纯林，阴坡非钙化生境为岷江冷杉－川滇长尾槭混交林。不同生境乔木层郁闭度、乔木密度、树高结构、直径结构均存在差异。 (2)钙化滩生境发现灌木41 种，平均盖度为18.49±1.72(％)，平均高度为52.12±4.45(cm)，优势种为直穗小檗(Berberis dasystachya)；阴坡非钙化生境发现灌木30 种，平均盖度为29.33±2.56 (％)，平均高度为119.55±8.01 (cm)，优势种为箭竹 (Fargesia spathacea) 、唐古特忍冬(Lonicera tangutica) 和袋花忍冬(Lonicera saccata)；半阳坡非钙化生境发现灌木29 种，平均盖度为31.35±1.93 (％)，平均高度为107.55±4.24 (cm)，优势种为箭竹(Fargesia spathacea)。不同生境灌木层结构和物种组成多样性差异显著，钙化滩生境的灌木盖度、高度总体上较非钙化的坡地生境低, 钙化滩生境灌木以小型叶的落叶灌木为主，沟两侧非钙化的坡地生境上则发育了丰富箭竹。 (3)钙化滩生境发现草本46 种，平均盖度为7.18±0.79 (％)，平均高度为5.04±0.26(cm)，以山酢浆草(Oxalis griffithii)为优势种；阴坡非钙化生境发现草本物种71 种，平均盖度达29.04±2.31(％)，平均高度为9.08±0.52(cm)，以钝叶楼梯草(Elatostema obtusum)、山酢浆草为优势种；半阳坡非钙化生境草本物种50 种，平均盖度为以8.79±0.82(％)，平均高度为7.67±0.43 (cm)，以扇叶铁线蕨(Adiantum flabellulatum)、双花堇菜(Viola biflora)、华中蛾眉蕨(Lunathyrium shennongense)、山酢浆草为优势种。阴坡非钙化生境草本层片发育良好，多样性最为丰富，盖度和物种丰富度均显著高于钙化滩生境和半阳坡非钙化生境。 (4)钙化滩生境发现苔藓物种140 种，平均盖度达84.25±1.30 (％)，以仰叶星塔藓(Hylocomiastrum umbratum) 等大型藓类为优势种；阴坡非钙化生境发现苔藓物种115 种，平均盖度为79.29±1.64 (％)，以刺叶提灯藓(Mnium spinosum)、大羽藓(Thuidium cymbifolium)、毛尖燕尾藓(Bryhnia trichomitra)等个体较小的物种为优势种；半阳坡非钙化生境发现苔藓物种91 种，平均盖度为60.64±1.93 (％)，也以刺叶提灯藓为优势种。 (5)钙化滩生境、阴坡非钙化生境、半阳坡非钙化生境的物种数分别为234 种、221 种、175 种。乔木层的Shannon-Wiener 指数分别为0.75 ±0.12、1.87±0.12、1.78±0.07（灌木层，0.44±0.08、1.71± 0.15、2.49±0.06；草本层，0.33±0.13、1.31±0.15 、2.15±0.08； 苔藓层1.30±0.11、2.08±0.04、1.73±0.11，）；Pielou 均匀度指数分别为0.45±0.05、0.29±0.06、0.28±0.08（灌木层，0.75±0.03、0.68±0.05、0.52±0.06；草本层，0.68±0.02、0.77±0.02、0.74±0.02；苔藓层，0.40±0.03、0.63±0.02、0.52±0.03）；Simpson's 优势度指数分别为0.63±0.06、0.78±0.04、0.83±0.07（灌木层，0.21±0.03、0.28±0.05、0.45±0.06；草本层，0.25±0.02、0.12±0.01、0.17±0.01；苔藓层，0.45±0.04、0.18±0.01、0.31±0.04）。三种生境间乔木层、草本层的Sorenson 群落相似性系数较低, 灌木层、苔藓层的的Sorenson 群落相似性系数较高。 综上所述，黄龙岷江冷杉林的群落结构、植物多样性在三种生境间存在差异性，这将意味着我们在进行黄龙世界自然遗产地的森林经营管理时要较多地关注岷江冷山林群落在不同生境中的差异性。 There were multiplex habitat types, complicated community structure and abundant species composition in the Huanglong World Natural Heritage Site. Uncovering the differences of biodiversity among different habitats was a precondition to understand the distribution, formation and sustaining mechanism of the biodiversity, and the foundation of biodiversity conservation. In the present study, using plenty of quadrants, we investigated the community structure and the biodiversity of the primitive Abies faxoniana forest in different habitats (travertine bottomland, semi-sunny-slope non-calcified habitat and shady-slope non-calcified habitat) in the Huanglong World Natural Heritage Site. The main results are as follows: All the primitive Abies faxoniana forests in the three habitats were uneven-aged with obvious vertical structure including tree layer, shrub layer, herb layer and bryophyte layer. A total of 386 higher plants including 163 vascular plant species (103 generic, 46 families) and 223 bryophyte species (83 generic, 38 families) were investigated. The structure and species composition of each layer are as follows: (1) There were 18, 13 and 8 tree species in travertine bottomland, shady-slope non-calcified habitat and semi-sunny-slope non-calcified habitat, respectively. The tree layers in all habitats can be divided into two clear sub-layers. The upper tree layers were dominated by Abies faxoniana, and the lower tree layers were dominated by uneven-aged Abies faxoniana or other phanerophytes species. There were Abies fargesii , Picea asperata and Betula platyphylla besides the dominated species (Abies faxoniana) in the upper tree layer in travertine bottomland, and the lower tree layers were dominated by uneven-aged Abies faxoniana; There were Abies fargesii and Betula platyphylla besides the dominated species (Abies faxoniana) in the upper tree layer in shady-slope non-calcified habitat, and the lower tree layers were dominated by Acer caudatum var. prattii; There was Abies fargesii besides the dominated species (Abies faxoniana) in the upper tree layer semi-sunny-slope non-calcified habitat, and the lower tree layers were dominated by uneven-aged Abies faxoniana. According to composition percentage of dominate species in tree layer, both the forest in travertine bottomland and in semi-sunny-slope non-calcified habitat could be ranked as pure forest, and the forest in shady-slope non-calcified habitat could be ranked as mingled forest. There were significant differences in crown density, plant density, height structure and diameter structure among the three habitats. (2) A total of 41 shrub species (average coverage 18.49±1.72％; average height 52.12±4.45 ㎝)were found in travertine bottomland, and the dominate species was Berberis dasystachya; A total of 30 shrub species (average coverage 29.33±2.56 ％;average height 119.55±8.01 ㎝)were found in shady-slope non-calcified habitat, and the dominate species was Fargesia spathacea, Lonicera tangutica and Lonicera saccata. A total of 29 shrub species (average coverage 31.35±1.93％; average height 107.55±4.24 ㎝) were found in semi-sunny-slope non-calcified habitat, and the dominate species was Fargesia spathacea. There were significant differences in structure and species diversity of the shrub layers among the three habitats. The coverage and height of shrub had lower value in travertine bottomland than in two non-calcified habitats. Moreover, travertine bottomland was dominated by deciduous shrub species with microphyll and non-calcified habitats developed abundant Fargesia spathacea species. (3) A total of 46 herb species (average coverage 7.18±0.79％;average height 5.04±0.26 ㎝)were found in travertine bottomland, and the dominate species was Oxalis griffithii; A total of 71 herb species (average coverage 29.04±2.31％;average height 9.08±0.52 ㎝)were found in shady-slope non-calcified habitat, and the dominate species was Elatostema obtusum and Oxalis griffithii. A total of 50 herb species (average coverage 8.79±0.82％;average height 7.67±0.43 ㎝) were found in semi-sunny-slope non-calcified habitat, and the dominate species was Adiantum flabellulatum, Viola biflora, Lunathyrium shennongense and Oxalis griffithii. Herb layers developed well in shady-slope non-calcified habitat and had the higher species richness and coverage than travertine bottomland and semi-sunny-slope non-calcified habitat. (4) A total of 140 bryophyte species (average coverage 84.25±1.30％)were found in travertine bottomland, and the dominate species was big bryophyte species such as Hylocomiastrum umbratum and so on; A total of 115 bryophyte species (average coverage 79.29±1.64％)were found in shady-slope non-calcified habitat, and the dominate species was small bryophyte species such as Mnium spinosum, Thuidium cymbifolium, Bryhnia trichomitra and so on. A total of 91 bryophyte species (average coverage 60.64±1.93％) were found in semi-sunny-slope non-calcified habitat, and the dominate species was Mnium spinosum. (5) There were 234, 221 and 175 plant species in travertine bottomland, shady-slope non-calcified habitat and semi-sunny-slope non-calcified habitat, respectively. Shannon-Wiener index of the tree layer was 0.75 ±0.12, 1.87±0.12 and 1.78±0.07 (the shrub layer, 0.44±0.08, 1.71± 0.15 and 2.49±0.06; the herb layer, 0.33±0.13, 1.31±0.15 and 2.15±0.08; the bryophyte layer, 1.30±0.11, 2.08±0.04 and 1.73±0.11.) for the three habitats, respectively; Pielou index of the tree layer was 0.45±0.05, 0.29±0.06 and 0.28±0.08 (the shrub layer, 0.75±0.03, 0.68±0.05 and 0.52±0.06; the herb layer, 0.68±0.02, 0.77±0.02 and 0.74±0.02; the bryophyte layer, 0.40±0.03, 0.63±0.02 and 0.52±0.03.) for the three habitats, respectively. Simpson's index of the tree layer was 0.63±0.06, 0.78±0.04 and 0.83±0.07 (the shrub layer, 0.21±0.03、0.28±0.05、0.45±0.06; the herb layer, 0.25±0.02, 0.12±0.01 and 0.17±0.01; the bryophyte layer, 0.45±0.04, 0.18±0.01 and 0.31±0.04.) for the three habitats, respectively. There were low Sorenson index both in the tree layer and in the herb layer among the three habitats, whereas, high Sorenson index occurred both in the shrub layer and in the bryophyte layer. To sum up, there were differences both in community structure and plant diversity among the three different habitats, which means that we should pay more attention to habitats heterogeneities of the primitive Abies faxoniana forest when we take action to manage the forest in the Huanglong World Natural Heritage Site.

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.