番鸭cDNA-AFLP体系的建立

以番鸭脑垂体为材料,用Trizol法提取总RNA,用Superscipt Ⅱ-RT合成第一链cDNA,RNase H和DNA Polymerase合成第二链cDNA,最后用EcoR Ⅰ/Mse Ⅰ分步酶切cDNA各2h,经T4连接酶合成双链cDNA,预扩增反应后产物稀释40倍进行选择性扩增反应.经1％琼脂糖电泳及6％变...     

策略构建小黑杨茎形成层全长cDNA文库

为了研究小黑杨木材形成过程中的关键基因,以2年生小黑杨茎形成层组织为试验材料提取RNA,采用SMART (RNA 转录过程中的5′末端转换机制) 技术合成cDNA第一链,通过LD-PCR合成双链cDNA。利用SfiⅠ限制性酶酶切后将其连接到质粒载体pDNR-LIB上,采用电穿孔法将重组质粒转化到大肠杆菌感受态细胞DH5α中,构建全长cDNA文库。文库质量鉴定表明:原始文库滴度为2.18×106 pfu/mL,扩增后的文库滴度为5.46×109 pfu/mL,重组率为96%。插入片段长度在0.5～2.0 kb之间,平均长度为1.12 kb,表明构建的小黑杨茎形成层cDNA文库较为理想。      

Structure of ribonuclease H phased at 2 A resolution by MAD analysis of the selenomethionyl protein

Ribonuclease H digests the RNA strand of duplex RNA.DNA hybrids into oligonucleotides. This activity is indispensable for retroviral infection and is involved in bacterial replication. The ribonuclease H from Escherichia coli is homologous with the retroviral proteins. The crystal structure of the E. coli enzyme reveals a distinctive alpha-beta tertiary fold. Analysis of the molecular model implicates a carboxyl triad in the catalytic mechanism and suggests a likely mode for the binding of RNA.DNA substrates. The structure was determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the recombinant selenomethionyl protein.     

SMART策略构建小黑杨茎形成层全长cDNA文库

为了研究小黑杨木材形成过程中的关键基因,以2年生小黑杨茎形成层组织为试验材料提取RNA,采用SMART(RNA转录过程中的5′末端转换机制)技术合成cDNA第一链,通过LD-PCR合成双链cDNA。利用SfiⅠ限制性酶酶切后将其连接到质粒载体pDNR-LIB上,采用电穿孔法将重组质粒转化到大肠杆菌感受态细胞DH5α中,构建全长cDNA文库。文库质量鉴定表明:原始文库滴度为2.18×106pfu/mL,扩增后的文库滴度为5.46×109pfu/mL,重组率为96%。插入片段长度在0.5～2.0kb之间,平均长度为1.12kb,表明构建的小黑杨茎形成层cDNA文库较为理想。     

欧洲油菜花粉cDNA文库的构建

从欧洲油菜（Ｂｒａｓｓｉｃａｎａｐｕｓ）成熟花粉中提纯出ｍＲＮＡ．ｐｏｌｙ（Ａ）＋ｍＲＮＡ在ｏｌｉｇｏ（ｄＴ）引导下，用反转录酶合成ｃＤ－ＮＡ第一链，用大肠杆菌ＲＮＡ酶Ｈ和大肠杆菌ＤＮＡ聚合酶Ｉ置换合成ＣＤＮＡ第二链．加上ＥｃｏＲＩ／ＮｏｔＩ接头。最后将双链ＤＮＡ克隆子λｇｔｌｌ载体中。用Ｂｃｐｌ作探针，杂交筛选构建的ｃＤＮＡ文库。     

Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template

Full-length poliovirus complementary DNA (cDNA) was synthesized by assembling oligonucleotides of plus and minus strand polarity. The synthetic poliovirus cDNA was transcribed by RNA polymerase into viral RNA, which translated and replicated in a cell-free extract, resulting in the de novo synthesis of infectious poliovirus. Experiments in tissue culture using neutralizing antibodies and CD155 receptor-specific antibodies and neurovirulence tests in CD155 transgenic mice confirmed that the synthetic virus had biochemical and pathogenic characteristics of poliovirus. Our results show that it is possible to synthesize an infectious agent by in vitro chemical-biochemical means solely by following instructions from a written sequence.     

草莓果实cDNA文库的构建及鉴定

草莓是研究果实发育的模式植物。基因表达谱分析已成为目前分子生物学领域重要的研究方向。草莓cDNA文库构建是研究果实发育基因表达的基础工作。本研究从草莓白果中提取高质量的总RNA,通过MMLV反转录酶的作用,把RNA中的mRNA反转成cDNA第一链,再利用特异引物通过LD-PCR扩增合成双链cDNA。将纯化带有黏性末端的双链cDNA与λTrip I Ex2载体进行连接,最后对重组载体进行E.coli XL1-Blue体外包埋为cDNA原始文库。经过鉴定,原始文库的滴度为1.13×10-7 cfu/mL,重组率为100%,插入片段多分布在0.2~2.0kb之间,表明构建的草莓果实cDNA文库质量较高,能够满足后续的分子生物学研究工作的需要,同时也为其他果树相关研究提供借鉴。     

The structural basis of ribosome activity in peptide bond synthesis

Using the atomic structures of the large ribosomal subunit from Haloarcula marismortui and its complexes with two substrate analogs, we establish that the ribosome is a ribozyme and address the catalytic properties of its all-RNA active site. Both substrate analogs are contacted exclusively by conserved ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no protein side-chain atoms closer than about 18 angstroms to the peptide bond being synthesized. The mechanism of peptide bond synthesis appears to resemble the reverse of the acylation step in serine proteases, with the base of A2486 (A2451 in Escherichia coli) playing the same general base role as histidine-57 in chymotrypsin. The unusual pK(a) (where K(a) is the acid dissociation constant) required for A2486 to perform this function may derive in part from its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a buried phosphate that could stabilize unusual tautomers of these two bases. The polypeptide exit tunnel is largely formed by RNA but has significant contributions from proteins L4, L22, and L39e, and its exit is encircled by proteins L19, L22, L23, L24, L29, and L31e.     

巴西橡胶树胶乳酵母双杂交cDNA表达文库的构建及分析

提取巴西橡胶树胶乳总RNA,并纯化mRNA.采用特异引物（oligo-dT）反转出第一链DNA,经LDPCR合成第二链DNA.将ds cDNA和经Sma Ⅰ线性化的pGADT7-Rec质粒共转化Y187酵母菌,构建酵母双杂交cDNA表达文库.结果表明,转化单菌落个数为2.8 ×107;文库滴度为4.92×107·mL-1;插入片段大小主要分布于500 ～2 000 bp间;重组率为96％.实验数据表明该文库能满足后续的杂交筛选.     

大丽轮枝菌毒素诱导表达陆地棉cDNA序列的克隆与定位

 为获得陆地棉黄萎病抗性相关候选基因，利用棉花黄萎病菌大丽轮枝菌V991毒素粗提物诱导耐黄萎病陆地棉品种'中棉12'应激基因的表达。在诱导幼根24 h时间点提取总RNA，PCR法合成双链cDNA，应用抑制消减杂交技术[1]，得到了差异表达的180个克隆。经反Northern blot筛选出15个受毒素粗提物诱导应激表达的阳性cDNA克隆，将其命名为Vdrg（response gene induced by toxin of Verticillium dahliae）。本研究对所得阳性cDNA克隆进行了序列分析，并利用Pima S6 与 Acala Maxxa 海陆杂交四倍体棉花F2群体对Vdrg序列进行了棉花基因组定位。     

水稻条纹病毒楚雄分离物一个重组RNA序列分析

为检测水稻条纹病毒(Rice stripe virus,RSV)基因组存在的重组RNA,采用RT-PCR方法扩增并克隆云南楚雄分离物(YCX07)的RNA重组片段YCX07R.克隆重组片段并测定序列.序列分析表明,YCX07R由RSVRNA2的3''端序列与RNA3的3''端序列重组而成,具有RSV基因组结构特征,采取双义编码策略,即在正、负链的5''端分别存在一个阅读框,编码两个重组蛋白YCX07R-5P、YCX07R-3P.分析推测重组更可能通过类似于脊髓灰质炎病毒(Poliovirus)的引物互补延伸机制发生.     

单链环状DNA添加剂抑制基因表达序列分析Ditag聚合酶链反应引物多聚体生成

[目的]研究利用单链环状DNA抑制聚合酶链反应（PCR）副产物的作用效果。[方法]设计并合成单链环状DNA,并以具同样序列发卡状结构的单链DNA为对照组,在基因表达序列分析（SAGE）Ditag PCR反应中加入上述不同浓度的DNA,利用电泳技术检测不同结构（环状/单链）以及不同浓度单链DNA对PCR多聚体附产物的抑制作用。[结果]发卡状结构的单链DNA无法抑制PCR引物多聚体副产物的产生;而在70～150 nmol/L终浓度范围内,单链环状DNA可以抑制SAGE Ditag PCR反应中引物多聚体的生成,并且不影响SAGE不同tag间的表达丰度差异。[结论]单链环状DNA可以作为PCR反应中引物多聚体生成的有效抑制剂。     

A protein sensor for siRNA asymmetry

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.     

Chemical etiology of nucleic acid structure: the alpha-threofuranosyl-(3'-->2') oligonucleotide system

TNAs [(L)-alpha-threofuranosyl oligonucleotides] containing vicinally connected (3'-->2') phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. Being derived from a sugar containing only four carbons, TNA is structurally the simplest of all potentially natural oligonucleotide-type nucleic acid alternatives studied thus far. This, along with the base-pairing properties of TNA, warrants close scrutiny of the system in the context of the problem of RNA's origin.     

Rapid switching of plant gene expression induced by fungal elicitor

The pattern of messenger RNA synthesis in suspension-cultured bean cells (Phaseolus vulgaris L.) was analyzed by blot hybridization and in vitro translation of newly synthesized messenger RNA. The RNA was separated from preexisting RNA by organomercurial affinity chromatography after in vivo labeling with 4-thiouridine. The elicitor induced the synthesis of messenger RNA's encoding phenylalanine ammonia-lyase, chalcone synthase, and chalcone isomerase, three enzymes of phenylpropanoid metabolism involved in the synthesis of isoflavonoidderived phytoalexins. This is part of a rapid and extensive change in the pattern of messenger RNA synthesis directing production of a set of proteins associated with expression of disease resistance.