Guanosine Diphosphate Mannose in Conformational Relation to Other Suger Nucleotides and Phosphates in Solution

Molecular conformational transition of GDPMan and solution conformation of α-d- mannopyranose moiety in Man-l-P and GDPMan were examined in relation to other sugar nucleotides and phosphates. GDPMan and other sugar nucleotides examined revealed changes in the optical rotation in sigmoidal curve in water by addition of urea. The change was reversible without significant decomposition and is attributable to dissociation of an ordered form into a random form. Optical conformational values in 8m urea solution were+116° for GDPMan, +58°~+79° for UDPGlc, +79° for UDPGal, +135°~+143° for UDPGlcNAc, and +138°~ +155° for UDPGIcA.

NMR analysis and periodate oxidation study revealed the ⁴C₁ conformation of α-d-hexopyranose moieties in Man-1-P, Glc-l-P, GDPMan, UDPGlcNAc and UDPGalNAc.     

Studies on Abscisic Acid

Methyl α-ionylideneacetates were oxidized with selenium dioxide to a mixture of methyl 3′-keto-β-ionylideneacetates and a small amount of methyl 4′-keto-α-ionylidene-acetates followed by treatment with active manganese dioxide. By a similar oxidation methyl 3′-keto-β-ionylideneacetates were prepared from methyl β-ionylidene acetates. Methyl 4′-keto-α-ionylideneacetates were obtained by oxidation of methyl α-ionylideneacetates with tert-butyl chromate. Dehydrobromination of methyl bromoionylideneacetate, obtained by bromination of methyl 2-trans-α-ionylideneacetate with N-bromosuccinimide, gave a mixture of methyl 2-trans-dehydro-β-ionylideneacetate and methyl 2-cis-dehydro-β-ionylideneacetate. The growth inhibitory activities of these sesquiterpene carboxylic acids and keto esters on rice seedlings were tested.     

Design,construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well‐established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin‐target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase‐7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.     

锌胁迫下小麦SSH文库的构建及初步分析

Zn是植物必需的微量元素,同时也是近年来造成环境污染的重金属元素之一.为了从基因表达水平揭示小麦Zn胁迫响应的分子机制,本研究利用抑制差减杂交(suppression subtractive hybridization,SSH)技术构建了Zn胁迫（0.5 mmol/L,1 mmol/L）下小麦的正反向SSH文库.从正反向文库中随机挑选阳性单克隆,并利用通用引物T7/Sp6对其进行验证. 结果显示,正反库中分别获得307和821个EST序列,其片段长度在200～1 000 bp之间,它们反映了Zn胁迫下特异响应的基因.利用BLASTn和BLASTx将这些EST序列进行比对分析,在正、反库中分别筛选出221和641个uniESTs,其中751个uniESTs被注释（包括正库中193个和反库中558个）.这些序列的功能主要涉及信号转导、抗氧化防御、转录与翻译、物质运输、核糖体结构、能量代谢,以及一些功能未知的基因.     

水稻二化螟和三化螟基因组ddRADseq文库的构建

本文介绍了构建水稻二化螟和三化螟"双酶切限制性酶切位点关联DNA测序"(Double digest restrictionsite associated DNA sequencing,ddRADseq)文库的方法。利用安捷伦2100生物分析仪对4种单酶切及2种双酶切的酶切产物片段大小及分布范围进行分析,筛选出Mlu C I和Nla III两种限制性内切酶组合对螟虫基因组DNA进行酶切。酶切后的DNA片段两端连接上特定的P1、P2接头后,用Pippin Prep回收大小为285-435 bp的DNA片段。通过PCR扩增进行文库的富集并引入index序列。构建好的ddRADseq文库用琼脂糖凝胶电泳和生物分析仪进行质量检测。本方法所构建的文库DNA片段长度、分布和摩尔浓度能够达到Illumina平台测序的技术要求。本研究证实了利用Mlu C I和Nla III组合酶切构建水稻螟虫基因组ddRADseq文库的可行性,为在水稻螟虫中利用ddRADseq技术开展生物地理学、种群遗传学和系统发育重建等方面的研究奠定基础。     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。