Construction, characterization and chromosomal mapping of bacterial artificial chromosome (BAC) library of Yunnan snub-nosed monkey (Rhinopithecus bieti)

We constructed a high redundancy bacterial artificial chromosome library of a seriously endangered Old World Monkey, the Yunnan snub-nosed monkey (Rhinopithecus bieti) from China. This library contains a total of 136 320 BAC clones. The average insert size of BAC clones was estimated to be 148 kb. The percentage of small inserts (50-100 kb) is 2.74%, and only 2.67% non-recombinant clones were observed. Assuming a similar genome size with closely related primate species, the Yunnan snub-nosed monkey BAC library has at least six times the genome coverage. By end sequencing of randomly selected BAC clones, we generated 201 sequence tags for the library. A total of 139 end-sequenced BAC clones were mapped onto the chromosomes of Yunnan snub-nosed monkey by fluorescence in-situ hybridization, demonstrating a high degree of synteny conservation between humans and Yunnan snub-nosed monkeys. Blast search against human genome showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the BAC library. This library and the mapped BAC clones will serve as a valuable resource in comparative genomics studies and large-scale genome sequencing of nonhuman primates. The DNA sequence data reported in this paper were deposited in GenBank and assigned the accession number CG891489-CG891703.

Anti-HER-2-SEC2融合免疫毒素的构建和活性研究

以PCR技术从金黄色葡萄球菌基因组DNA中首次克隆编码成熟SECZ蛋白的全基因sec2。该基因共717bp，编码239个氨基酸，Genbank Accession number：AY450554。构建了SEC2的表达载体pET-28a-sec2，并在大肠杆菌BL21（DE3）中高效表达可溶性rSEC2蛋白。经亲和层析纯化，其纯度在95％以上，平均回收量为每升培养物40mg。纯化的rSEcZ保持了与野生型相当的生物学活性。以限制性核酸内切酶连接技术分别将两个抗人表皮生长因子受体HER-2单链抗体基因通过DNA Linker与sec2融合，构建融合基因b-l-sec2和ml小sec2，并以两种方式表达纯化。以pET-32a表达载体在E，coliAD494（DE3）中以氨基端融合大肠杆菌硫氧还蛋白（TrxA）形式高效表达融合蛋白TRX-B-L-SEC2和TRX-ML-L-SEC2，经亲和层析纯化，并以肠激酶切割得到成熟融合免疫毒素B-L-SEC2和ML-L-SEC2，其纯度在95％以上，平均回收量为每升培养物smg；以构建的新型表达载体pASK-75-EX在E.coliBL21（ED3）中以不溶性包涵体形式表达融合免疫毒素蛋白，经变性、纯化和复性后得到具有生物学活性的融合免疫毒素，其纯度在95％以上，平均回收量为每升培养物30mg。以两种方式制备的融合免疫毒素都保持了SECZ蛋白的免疫原性，都能有效刺激人外周血单个核细胞的增殖，并且都显示出在体外与HER-2过表达的乳腺癌细胞SK-Br-3特异性结合能力，具有显著的靶向性抑瘤作用。用PcR方法扩增了编码TrxA蛋白的基因trxA并克隆至表达载体pET-28a启动子上游，构建了一种在单质粒中利用两个相同的启动子游离共表达硫氧还蛋白与目的蛋白的表达载体。利用该载体可使TrxA与外源蛋白在大肠杆菌BL21（DE3）中以非融合形式高效共表达。共表达的TrxA可明显促进外源蛋白单链抗体ML3.9（scFv-ML）、3一轻基苯甲酸-6-单加氧酶（3HBA）的可溶性表达；并明显减少肠毒素C2（SEC2）、结核杆菌螺旋酶A亚基（GYRA）的包涵体表达。

青稞B组醇溶蛋白基因与上游调控区的克隆及基因表达

高等植物种子胚乳贮藏蛋白是种子发芽时的主要氮源，也是人类和动物食用植物蛋白的主要来源。大麦种子胚乳贮藏蛋白主要是醇溶蛋白（hordeins），占大麦胚乳总蛋白的50–60%。根据大麦醇溶蛋白的大小和组成特点，大麦醇溶蛋白被划分为三种类型：富硫蛋白亚类(B，γ-hordeins)、贫硫蛋白亚类（C-hordeins)以及高分子量蛋白亚类(D-hordeins)。B组和C组醇溶蛋白是大麦胚乳的两类主要贮藏蛋白，它们分别占大麦总醇溶蛋白成分的70–80%和10–12%。遗传分析表明，大麦B、C、D和γ-组醇溶蛋白分别是由位于大麦第五染色体1H（5）上的Hor2、Hor1、Hor3和Hor5位点编码。Hor2位点编码大量分子量相同但组成不同的B组醇溶蛋白（B-hordein）。B-hordein的种类、数量和分布是影响大麦酿造、食用及饲养品质的重要因素之一。为深入了解B-hordein基因家族的结构和染色体组织，探明Hor2位点基因表达的发育调控机制，最终达到改良禾谷类作物籽粒品质的目的，本研究以青藏高原青稞为材料，采用同源克隆法，分别克隆B-hordein基因和启动子，通过原核生物表达验证B-hordein基因功能，并利用实时定量PCR探索B-hordein基因表达时空关系，取得如下研究结果： 1. 以具有特殊B组醇溶蛋白亚基组成的9份青藏高原青稞为材料，根据GenBank中三个B-hordein基因序列（GenBank No. X03103, X53690和X53691）设计一对引物，通过PCR扩增，获得23个B-hordein基因克隆并对其进行了序列分析。核苷酸序列分析表明，所有克隆均包含完整的开放阅读框。有11个克隆都存在一个框内终止密码子，推测这11个克隆可能是假基因。推测的氨基酸序列分析表明，所有大麦B-hordein具有相似的蛋白质基本结构，均包括一个高度保守的信号肽、中间重复区以及C-端结构域。不同大麦种重复区内重复基元的数目有较大差异。青稞材料Z07–2和Z26的B-hordeins仅具有12个重复基元结构，更接近于野生大麦。这些重复基元数目的差异导致了重复区序列长度和结构的变异。这种现象极可能是由于醇溶谷蛋白基因在进化过程中染色体的不平衡交换或复制滑动所造成的。对所克隆基因和禾本科代表性醇溶谷蛋白基因进行聚类分析，结果表明所有来自栽培大麦的B-hordeins聚类成一个亚家族，来自野生大麦的B-hordeins以及普通小麦的LMW-GS聚类成另外一个亚家族，表明这两个亚家族的成员存在显著差异。此外，我们发现B-hordein基因推测的C-末端序列具有一些有规律的特征：即具有相同C-末端序列的B-hordein基因在系统发生树中聚类为同一个亚组（除BXQ053，BZ09-1，BZ26-5分别单独聚为一类外）。这个特征将有助于我们对所有B组醇溶蛋白基因家族成员进行分类，避免了在SDS-PAGE电泳图谱上仅依靠大小分类的局限性。 2. 根据上述克隆的青稞B-hordein基因的5’端序列设计三条基因特异的反向引物，以青稞Z09和Z26的基因组DNA为模板，采用SON-PCR和TAIL-PCR技术分离克隆出8个B-hordein基因的上游调控序列（命名为Z09P和Z26P）。序列分析表明，推测的TATA box位于–80 bp，CAAT–like box位于–140 bp处。此外，Z09P和Z26P中有六个序列在–300 bp处均存在一个由高度保守的EM基序和类GCN4基序构成的胚乳盒(Endosperm Box，EB)，在约–560 bp处存在一个胚乳盒类似结构。而Z09P-2和Z26P-3不存在保守的胚乳盒或其类似结构，预示着这两个启动子所调控的基因表达可能受不同类型反式作用因子的调节，推测该启动子对基因的表达调控具有多样性。 3. 将B-hordein基因的开放阅读框定向克隆到表达载体pET-30a中，将其导入大肠杆菌表达菌株BL21中进行外源基因的诱导表达以验证所克隆基因的功能。结果表明仅含重组子pET-BZ07-2和pET-BZ26-5的BL21细菌有目的表达蛋白产生。在诱导3 h时的蛋白表达量最高；3 mM IPTG诱导的蛋白表达量要高于1 mM IPTG诱导的表达量。这为分离纯化B-hordein蛋白以及进一步研究其对大麦籽粒品质的影响奠定基础。 4. 根据从青稞Z09和Z26中分离克隆的B-hordein基因序列设计一对基因特异的引物，同时，选择大麦α-微管蛋白基因（GenBank no. U40042）为看家基因并设计特异引物，利用实时荧光定量PCR检测了青稞籽粒4个胚乳发育时间段的B-hordein基因表达，荧光定量结果显示：两份材料中B-hordein基因的表达量均随发育过程的进行而逐渐升高。Z09中B-hordein基因在开花后7天开始转录，而Z26开花4天后就有低水平B-hordein的表达，这表明Z26中B-hordein基因可能比Z09表达的较早或者Z09中B-hordein基因表达水平较低以致于不能被检测到。此外，在4个不同的胚乳发育时期中，Z26中B-hordein基因的表达量均高于Z09材料。在开花12天到18天的过程中，Z09和Z26中B-hordein基因的表达水平有一个急剧性的升高。这说明在不同胚乳发育时期，Hor2位点的B-hordein等位基因变异体存在mRNA的差异表达。 Seed endosperm storage proteins in higher plants are the main resources of nitrogen for germinating and plant proteins for human and animals. Barley prolamins (also called hordeins) are the major storage proteins in the endosperm and account for 50–60% of total proteins. Hordeins are classically divided into three groups: sulphur-rich (B, γ-hordeins), sulphur-poor (C-hordeins) and high molecular weight (HMW, D-hordeins) hordeins based on the size and composition. B-hordeins and C-hordeins are two major groups and each respectively account for about 70-80% and 10-12% of the total hordein fraction in barley endosperm. Genetic analysis showed that B-, C-, C-, γ-hordeins are encoded by Hor2, Hor1, Hor3 and Hor5 locus on the chromosome 1H (5). Hor2 locus is rich in alleles that encode numerous heterogeneous B-hordein polypeptides. It is reported that B-hordein species, quantity and distribution are significant factors affecting malting, food and feed quality of barley. To understand comprehensively the structure and organization of B-hordein gene family in hull-less barley and explore the developmental control mechanisms of Hor2 locus gene expression and eventually to better exploitation in crop grain quality improvement, we isolated and cloned B-hordein genes and promotors of hull-less barley from Qinghai-Tibet Plateau by PCR, and testified their expression founction in bacteria expression system and explore their spatial and temporal expression pattern by quantitative real time PCR. Our results are as followed, 1. Twenty-three copies of B-hordein gene were cloned from nine hull-less barley cultivars of Qinghai-Tibet Plateau with special B-hordein subunits and molecularly characterized by PCR, based on three B-hordein genes published previously (GenBank No. X03103, X53690 and X53691). DNA sequences analyses confirmed that the six clones all contained a full-length coding region of the barley B-hordein genes. Eleven clones all contain an in-frame stop codon and they are probably pseudogenes. The analysis of deduced amino acid sequences of the genes shows that they have similar structures including signal peptide domain, central repetitive domain, and C-terminal domain. The number of the repeats was largerly variable and resulted in polypeptides in different sizes or structures among the genes. Twelve such repeated motifs were found in Z07–2 and Z26, and they are close to those of the wild barleys, and it is most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins. The relatedness of prolamin genes of barley and wheat was assessed in the phylogenetic tree based on their polypeptides comparison. Our phylogenetic analysis suggested that the predicted B-hordeins of cultivated barley formed a subfamily, while the B-hordeins of wild barleys and the two most similar sequences of LMW-GS of T. aestivum formed another subfamily. This result indicated that the members of the two subfamilys have a distinctive difference. In addition, we found the B-hordeins with identical C-terminal end sequences were clustered into a same subgroup (except BXQ053，BZ09-1 and BZ26-5 as a sole group, respectively), so we believe that B-hordein gene subfamilies possibly can be classified on the basis of the conserved C-terminal end sequences of predicted polypeptide and without the limit of SDS-PAGE protein banding patterns. 2. The specific primers were designed according to the published sequences of barley B-hordein genes from Z09 and Z26. Using total DNA isolated from them as the templates, eight clones (designated Z09Pand Z26P) of upstream sequences of the known B-hordein genes was obtained by TAIL-PCR and SON-PCR. Sequences analysis shows that the putative TATA box was present at position –80 bp and CAAT-like box at position –140 bp. Besides, a putative Endosperm Box including an Endosperm Motif (EM) and a GCN4-Like Motif was found at position –300 bp in six clones, and another Endosperm-like box was found at positon –560 bp. While the Endosperm Box or Endosperm-like box was not found in Z09P-2 and Z26P-3. This may indicate that gene expression drived by the two promtors was probably controlled by different trans-acting factors and the genetic control mechanism of corresponding gene expression may be diverse. 3. The B-hordein genic region coding for the mature peptide was cloned into expression vector pET-30a and transformed into bacterial strain BL21 for identifying gene expression fountion. Protein SDS–PAGE analysis showed that only the transformed lysate with the pET-BZ07-2 and pET-BZ26-5 constructs produced proteins related to B-group hordeins of barley, and the mounts of proteins induced by 3 mM IPTG and 3 h were higher than other conditions. This established a base for isolating and putifying B-hordein and further exploring their effects on barley grain quality. 4. The gene-specific primers of B-hordein genes from Z09 and Z26 were used for the quantification of B-hordein gene expression. The α-tubulin gene from Hordeum vulgare subsp. vulgare (GenBank accession number U40042) was used as a control gene. The result shows the transcription of the B-hordein genes in Z09 was found 7 days after flowering, while the transcription of the B-hordein genes in Z26 was found 4 days after flowering, but at a very low level, and it suggested that the B-hordein genes in Z26 probably expressed earlier than those in Z09, or the B-hordein genes in Z09 expressed at so a lower level than Z26 that it can not detected. In addition, B-hordein genes in Z26 accession showed higher expression levels than those in Z09 in four developing stages. Furthermore, a progressive increase in the expression levels of the B-hordein genes between 12 and 18 days after anthesis was observed in both Z09 and Z26. It implies that the B-hordein allelic variants encoded by Hor2 locus exist the differential expression in mRNA levels of during barley endosperm development.

小麦胚乳蛋白组分和Glu-B3位点基因核心编码区差异与小麦品质关系的研究

小麦加工品质改良已成为我国小麦育种的主要目标之一。特别是我国加入WTO以后，对小麦产品的质量提出了更高的要求，小麦品质改良的任务将更加艰巨和重要，小麦胚乳蛋白是影响小麦加工品质性状的重要因素。因此，深入了解小麦胚乳蛋白对加工品质性状的影响及其分子基础，为品质改良提供理论依据和科学指导，对加速我国小麦品质育种和优质小麦生产具有重要意义。本研究选用在麦谷蛋白5个基因位点(Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3)上均含不同等位基因的小麦品种99G45和京771及Pm97034和京771杂交F9代共164个麦谷蛋白纯合系，及228个中国推广普通小麦品种和高代育成品系为试材，研究了麦谷蛋白Glu-1和Glu-3位点基因等位变异对籽粒蛋白、湿面筋含量、Zeleny沉降值和SDS沉降值间的关系；本研究还利用小麦A、B和D基因组中低分子量麦谷蛋白亚基（LMW-GS）基因特异引物，通过PCR方法克隆了1个Glu-A3位点和3个Glu-B3位点LMW-GS基因片段，在此基础上分析了不同等位基因对品质造成差异的分子基础；另外，本研究对中国近年推广的部分品种和育成的高代品系资源的多样性进行了分析。现将主要研究结果简述如下： 1. 对来自三个麦区的148份材料的醇溶蛋白组成进行了分析，结果表明，各麦区醇溶蛋白模式具有较大差异。在ω区,A7、B、E、F、G、J、P、Q、S和U仅存在于西南秋播麦区；A3、M、N、R、W和X仅存在于黄淮特种麦区；K仅存在于北方冬麦区；A6是北方冬麦区出现频率最高的带型模式，而西南秋播麦区中D出现的频率最高。ω-区的E、H和M几种模式是以前国内外未曾报道的。且初步确定，这些模式对品质性状具有正效应。至于γ区,A、B、D、E和F在各区均有出现，其中B和E在各区出现的频率都很高，在26.1-39.6％之间。相反，H 仅出现在黄淮特种麦区，J仅限于西南秋播麦区。对于β-区醇溶蛋白，B型模式在所有区中都相当高，而模式A仅存在于第三区.对于α-区，模式A在Ⅲ区而模式D在Ⅱ区出现的频率很高。1BL.1RS易位系在中国小麦品种中出现频率高达41.2％，在I, II和Ⅲ麦区的出现频率分别为 45.5、43.5和35.2%。各生态区模式的差异可能是品种适应不同生态条件和人为选择的结果，但这有待进一步证明。由于醇溶蛋白位点（Gli-1）与LMW-GS位点（Glu-3）紧密连锁，本结果可为下面确定普通小麦LMW-GS等位基因变异所用。 2. 利用Gli-1与Glu-3的紧密连锁，以228个小麦品种/系为材料，首次对中国小麦品种麦谷蛋白亚基的6个位点进行综合分析，研究小麦籽粒蛋白与品质性状间的关系，结果表明6个高分子量（HMW）和低分子量（LMW）麦谷蛋白位点对蛋白质含量的效应大小为，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3；对GMP含量的效应大小为, Glu-A3>Glu-B3>Glu-D1> Glu-B1>Glu-A1>Glu-D3；对湿面筋含量的效应大小为, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1；对Zeleny沉降值的效应大小为, Glu-A1> Glu-B3>Glu-D3>Glu-D1>Glu-B1>Glu-A3；对SDS沉降值的效应大小为, Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1。对蛋白含量而言，各位点的最佳组合方式为1、17+18、5+10、Glu-A3e、Glu-B3g、Glu-D3b；对湿面筋含量而言，各位点的最佳组合方式为1、6+8、5+10、Glu-A3d、Glu-B3c、Glu-D3b；对Zeleny沉降值而言，各位点的最佳组合方式为N、17+18、5+10、Glu-A3d、Glu-B3d、Glu-D3b；对SDS沉降值而言，各位点的最佳组合方式为1、7+8、2.2+12、Glu-A3b、Glu-B3g、Glu-D3b。另外，分析了稀有亚基对5+12与2.2+12与品质性状的关系，认为5+12对品质有负效应，2.2+12对品质有正效应。在品质育种时，应对优异组合或优异亚基加以利用。 3. 首次利用重组自交系（RILs）为材料，研究麦谷蛋白亚基表达量与品质性状的关系，通过对重组自交系中各HMW-GS表达量的分析，认为，就单个亚基的表达量而言，7亚基最高；其次为2亚基、5亚基、12亚基和10亚基；亚基9和1的表达量最小；N亚基不表达。对成对出现的亚基对而言，x型和y型亚基的总表达量2+12>5+10>7+9>17+18。就单个亚基与品质性状的关系而言，仅有10亚基的表达量与蛋白含量的相关性达5%的显著水平，2亚基的表达量与湿面筋含量呈负相关，显著水平也达5%，其余单个亚基对品质性状均无显著影响；就x型/y型亚基的比例来看，2/12和5/10对湿面筋含量都有显著的负效应；对某一位点等位基因控制的亚基表达总量来看，2+12对SDS沉降值有显著负效应。另外，本研究得出：2＋12的亚基对的负效应主要体现在2亚基上，且在同一位点上，x型亚基的表达量大于y型。所以推导稀有亚基组合2+10很可能也是劣质亚基。 4. 以 Glu-A1、Glu-B1、Glu-D1、Glu-B3和Glu-D3作为5个因素对99G45/京771和Pm97034/京771杂交后代的蛋白质含量和SDS沉降值进行多因素方差分析。结果表明，Glu-A1和Glu-D3对蛋白含量的加性效应达5％显著水平；Glu-D1 * Glu-D3对蛋白质含量的互作效应也达5%显著水平；其余位点的加性和互作效应对蛋白质含量的影响均不显著。对SDS 沉降值而言，Glu-D1的加性效应最大,贡献率为4.2 % ,达1 %显著水平,其次是Glu-B1位点,贡献率为3.3% ,达5%显著水平。其余位点对SDS 沉降值的加性和互作效应均未达5%显著水平。总体而言, 各位点对蛋白含量的效应大小为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3；对SDS沉降值的效应大小为Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。Glu-D1和Glu-D3位点上等位基因变异对蛋白含量有显著或极显著影响，含Glu-D1d和Glu-D3 GD、Glu-D3 JD基因的株系分别比含Glu-D1a和Glu-D3 PD基因的株系有较高的蛋白含量；在该遗传背景下，麦谷蛋白各基因位点对蛋白含量的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9>17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。对SDS沉降值的效应大小依次排列为：Glu-A1位点1>N；Glu-B1位点7+9=17+18>14+15；Glu-D1位点5+10>2+12；Glu-B3位点GB>JB>PB；Glu-D3位点GB>JB>PB。所以，对蛋白含量和SDS沉降值均较好的组合为1，7+9，5+10，GB，GD。 5. 因为GB和PB对品质的效应有显著差异，选取LMW-GS位点特异扩增引物对京771、99G45和Pm97034的Glu-B3位点进行扩增，结果得到三个不一样的扩增片段（Genebank号为DQ539657－DQ539659），得到的基因片段与Genebank中已报道的同类序列高度同源。通过克隆片段组成的分析，发现对Pm97034的序列较京771和99G45段少一个7氨基酸的重复单元，这可能是它较另外两个片段对面筋强度影响小的主要原因；另外，在99G45的序列中，124位处出现L（亮氨酸）代替P（脯氨酸），158位处出现了T（苏氨酸）代换M(蛋氨酸)，这可能是99G45Glu-B3位点序列对SDS沉降值的效应显著优于Pm97034的原因。 6．通过对RILs各位点同普通小麦品种（系）各位点与品质关系的比较，发现对SDS沉降值的效应，各位点在不同研究材料中是不同的，普通小麦中：Glu-B3>Glu-A1=Glu-D1=Glu-A3>Glu-D3>Glu-B1，RILs中：Glu-D1>Glu-B1> Glu-D3>Glu-A1> Glu-B3。利用重组自交系材料（完全排除了1BL/1RS易位干扰）所得到的结果与Gupta and MacRitchie (1994)所得结论一致。进一步证实了1BL/1RS易位对小麦品质的重要影响。对蛋白含量而言，普通小麦品种（系）中，Glu-D1>Glu-B3>Glu-A1=Glu-B1> Glu-A3=Glu-D3，RILs中，Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3，和对SDS沉降值的效应一样，推断在非1BL/1RS易位的情况下，各位点对其效应应为Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3。 对同一位点的等位基因而言，普通小麦和重组自交系中Glu-A1和Glu-D1上的等位基因对品质性状的贡献是一致的，但Glu-B1上的等位基因对SDS沉降值的贡献发生了变化，普通小麦中17+18>7+9，RILs中7+9>17+18，这可能也是1BL/1RS造成的。 Baking quality improved is one of the main object of wheat bread in China. The overall objective of the present studies was to increase the understanding about protein quality in wheat, i.e. to make it possible to improve the production of wheat with desired quality for different end-uses. With the analysis of gluten protein in RILs, 99G45/Jing 771 and Pm97034/Jing, and 228 wheat cultivars or lines in China, the correlations between glutenin compositions and protein content, glutenin macropolymer(GMP), wet gluten content, Zeleny sedimentation value and SDS sedimentation value contentand breadmaking quality were studied. Also a rapid and efficient detection method of geneticpolymorphism at Glu-B3 loci in wheat was established using polymerase chain reaction(PCR).The results obtained were as follows: 1. Cultivated Chinese wheat germplasm has been a valuable genetic resource in international plant breeding. Patterns of gliadin among cultivated Chinese accessions are unknown, despite the proven value and potential novelty. The objective of this work was to analyse the diversity within improved Chinese wheat germplasm. The electrophoretic banding patterns of gliadin in common wheat cultivars and advanced lines were determined by acid-polyacrylamide gel electrophoresis. For 148 leading commercial cultivars and promising advanced lines used in our study, 48 patterns were identified, 29 corresponding to ω-gliadin, 9 to γ-gliadin, 5 to β-gliadin and 5 to α-gliadin. The most frequent patterns were A6 in ω; B in γ; B in β and A in the region of α. 116 band types appeared in the148 samples: 94 accessions had unique gliadin types, and 22 gliadin types while not unique were found in 54 accessions. The gliadin patterns of Chinese wheat cultivars and lines greatly differed from the patterns of wheat lines from other countries. Three patterns, E, J, H, M, N and O in the ω-zone had not previously been reported. Three wheat zones，the Northern Winter Wheat Region, the Yellow and Huai Valley River valleys Winter Wheat Region and the Southwestern Winter Wheat Region，in China showed different frequencies in their gliadin patterns. This information can be used to monitor genetic diversity with Chinese wheat germplasm. 2. To analyse the relationship between the loci and characteristics quality, we utilized the 228 cultivars/lines. The results showed that : For protein content, Glu-D1 >Glu-B3>Glu-A1=Glu-B1>Glu-A3=Glu-D3. For GMP content, Glu-A3>Glu-B3 >Glu-D1>Glu-B1>Glu-A1>Glu-D3. For wet gluten content, Glu-B1>Glu-B3= Glu-D3>Glu-A3>Glu-A1>Glu-D1. For Zeleny sedimentation value, Glu-A1>Glu-B3> Glu-D3>Glu-D1>Glu-B1>Glu-A3, For SDS sedimentation value, Glu-B3>Glu-A1= Glu-D1= lu-A3>Glu-D3>Glu-B1。For protein content, the best combination of 6 loci is (1,17+18,5+10,Glu-A3e, Glu-B3g,Glu-D3b). For wet gluten content, the best combination of 6 loci is (1,6+8,5+10,Glu-A3d,Glu-B3c,Glu-D3b). For Zeleny sedimentation value, the best combination of 6 loci is (N,17+18,5+10,Glu-A3d, Glu-B3d, Glu-D3b). For SDS sedimentation value, the best combination of 6 loci is(7+8,2.2+12,Glu-A3b, Glu-B3g,Glu-D3b)。Additional, we analysed the relationship between the subunits 5+12 and 2.2+12, think that 5+12 was negative for quality, 2.2+12 is postive for quality. It should be effective utilized. 3. It’s the first time to utilize RILs to study the relationship between subunits expression quantity and characteristics quality. The results showed that: For single subunit, the expression quantity of 7 is the highest. Then the 2, 5, 12 and 10. The expression of subunit 9 and 1 is the lowest. Subunit N is not expressed. For subunits, the expression quantity of x type and y type are 2+12>5+10>7+9>17+18. The significant relation of 5% only showed between the expression quantity of subunit 10 and protein content. The relationship between expression quantity of others and characteristic quality was not significant. For x type/ytype, 2/12 and 5/10 is negative relation insignificant level. For the subunit(s) in a loci, Only 2+12 effect SDS sedimentation value negative in significant level. 4. With RILs 99G45/Jing 771 and Pm97034/Jing 771, we found that: The effective of Glu-A1, Glu-D3 and Glu-D1 * Glu-D3 for protein content is significant at 5% level. The effect of other loci for protein wre not significant. For SDS sedimentation value, the effect of Glu-D1is the highest, which contribution is 4.2 % .Then the Glu-B1, contribution is 3.3%. The effect of other loci for SDS sedimentationvalue were not significant. In total, for protein content: Glu-D3 > Glu-A1 > Glu-D1>Glu-B1>Glu-B3; for SDS sedimentationvalue: Glu-D1>Glu-B1> Glu-D3>Glu-A1>Glu-B3. The effect of alleles in Glu-D1 and Glu-D3 loci are significant at 1% or 5%. In Glu-A1, 1>N; Glu-B1, 7+9>17+18>14+15; Glu-D, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. For SDS sedimentation, Glu-A1, 1>N; Glu-B1, 7+9=17+18>14+15; Glu-D1, 5+10>2+12; Glu-B3, GB>JB>PB; Glu-D3, GB>JB>PB. The best combinations for SDS sedimentation value is 1，7+9，5+10，GB，GD. 5. Because of the difference of GB and PB for SDS sedimentation value, we selected the specific primer for LMW-GS loci to amplified the Glu-B3 of Jing771, 99G45and Pm97034. We got 3 amplify fragment (Gene Bank accession number are DQ539657－DQ539659）. We found that the fragment of Pm97034 were deleted a repetitive 7 amino acid domain, which is perhaps the reason effect the gluten strength. Furthermore, in the position 124 of sequence 99G45, L has been replaced with P. Position 158, T replaced M, which may be the reason why the Glu-B3 locus of 99G45 is prefer to Pm97034 when refer to SDS sedimentation value. 6. Comparing the results of RILs and common wheat, we found that perhaps just the1BL/1RS made the difference of loci in different accession.

金黄色葡萄球菌肠毒素C2的基因克隆、表达及其生物学活性

利用PCR技术从金黄色葡萄球菌的基因组DNA中克隆SEC2全长基因,PCR产物与pGEM-T载体连接,经测序证实后进行亚克隆,构建其表达载体pET-28a-SEC2,在大肠杆菌BL21(DE3)中表达成熟重组蛋白(rSEC2),纯化rSEC2蛋白并对其生物学活性进行研究. 结果表明:成功克隆了SEC2全长基因,测序证实该基因共717 bp,编码239个氨基酸,与GenBank中收录的SEC2成熟蛋白质序列完全一致,SEC2基因登录GenBank(Accession number:AY450554);构建了SEC2的表达载体pET-28a-SEC2,并在大肠杆菌BL21(DE3)中得到高效可溶性表达,可溶性的rSEC2经Ni2+亲和层析纯化达到电泳纯,纯化的rSEC2蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被rSEC2刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用.

Cloning and Comparative Studies of Seaweed Trehalose-6-Phosphate Synthase Genes

The full-length cDNA sequence (3219 base pairs) of the trehalose-6-phosphate synthase gene of Porphyra yezoensis (PyTPS) was isolated by RACE-PCR and deposited in GenBank (NCBI) with the accession number AY729671. PyTPS encodes a protein of 908 amino acids before a stop codon, and has a calculated molecular mass of 101,591 Daltons. The PyTPS protein consists of a TPS domain in the N-terminus and a putative TPP domain at the C-terminus. Homology alignment for PyTPS and the TPS proteins from bacteria, yeast and higher plants indicated that the most closely related sequences to PyTPS were those from higher plants (OsTPS and AtTPS5), whereas the most distant sequence to PyTPS was from bacteria (EcOtsAB). Based on the identified sequence of the PyTPS gene, PCR primers were designed and used to amplify the TPS genes from nine other seaweed species. Sequences of the nine obtained TPS genes were deposited in GenBank (NCBI). All 10 TPS genes encoded peptides of 908 amino acids and the sequences were highly conserved both in nucleotide composition (>94%) and in amino acid composition (>96%). Unlike the TPS genes from some other plants, there was no intron in any of the 10 isolated seaweed TPS genes.

Identification of ribosomal proteins specific to higher eukaryotic organisms

This report describes the identification of a novel protein named PS1D (Genbank accession number ), which is composed of an S1-like RNA-binding domain, a (cysteine)x3-(histidine) CCCH-zinc finger, and a very basic carboxyl domain. PS1D is expressed as two isoforms, probably resulting from the alternative splicing of mRNA. The long PS1D isoform differs from the short one by the presence of 48 additional amino acids at its amino-terminal extremity. Analysis of PS1D subcellular distribution by cell fractionation reveals that this protein belongs to the core of the eukaryotic 60S ribosomal subunit. Interestingly, PS1D protein is a highly conserved protein among mammalians as murine, human, and simian PS1D homologues share more than 95% identity. In contrast, no homologous protein is found in lower eukaryotes such as yeast and Caenorhabditis elegans. These observations indicate that PS1D is the first eukaryotic ribosomal protein that is specific to higher eukaryotes.

Vasorelaxin: A Novel Arterial Smooth Muscle-Relaxing Eicosapeptide from the Skin Secretion of the Chinese Piebald Odorous Frog (Odorrana schmackeri)

The defensive skin secretions of amphibians are a rich resource for the discovery of novel, bioactive peptides. Here we report the identification of a novel vascular smooth muscle-relaxing peptide, named vasorelaxin, from the skin secretion of the Chinese piebald odorous frog, Odorrana schmackeri. Vasorelaxin consists of 20 amino acid residues, SRVVKCSGFRPGSPDSREFC, with a disulfide-bridge between Cys-6 and Cys-20. The structure of its biosynthetic precursor was deduced from cloned skin cDNA and consists of 67 amino acid residues encoding a single copy of vasorelaxin (vasorelaxin, accession number: HE860494). Synthetic vasorelaxin caused a profound relaxation of rat arterial smooth muscle with an EC50 of 6.76 nM.

Transmit and receive filter design for OFDM based WLAN systems

We analyze the effect of different pulse shaping filters on the orthogonal frequency division multiplexing (OFDM) based wireless local area network (LAN) systems in this paper. In particular, the performances of the square root raised cosine (RRC) pulses with different rolloff factors are evaluated and compared. This work provides some guidances on how to choose RRC pulses in practical WLAN systems, e.g., the selection of rolloff factor, truncation length, oversampling rate, quantization levels, etc.

Identification of benzimidazole resistance in Cladobotryum dendroides using a PCR-based method

Cladobotryutn dendroides, causal agent of cobweb disease of Agaricus bisporus, has become increasingly resistant to methylbenzimidazole carbamate (MBC) fungicides following the extensive use of MBC in cultivated mushroom production in Ireland. Of 38 isolates of C. dendroides obtained from Irish mushroom units, 34 were resistant to carbendazim. Primers based on conserved regions of the -tubulin gene were used to amplify and sequence a portion of the -tubulin gene in C. dendroides. A point mutation was detected at codon 50 in isolates resistant to benzimidazole fungicides, causing an amino acid substitution from tyrosine to cysteine. Species-specific PCR primers were designed to amplify the region of the -tubulin gene containing this substitution. The point mutation removed an Ace I restriction site in the -tubulin gene sequence of resistant isolates. Digestion of the PCR product with Ace I thus provides a rapid diagnostic test to differentiate sensitive and resistant isolates of this fungus. EMBL accession number: YI2256.

A review of dynamic Bayesian network techniques with applications in healthcare risk modelling

Coping with an ageing population is a major concern for healthcare organisations around the world. The average cost of hospital care is higher than social care for older and terminally ill patients. Moreover, the average cost of social care increases with the age of the patient. Therefore, it is important to make efficient and fair capacity planning which also incorporates patient centred outcomes. Predictive models can provide predictions which their accuracy can be understood and quantified. Predictive modelling can help patients and carers to get the appropriate support services, and allow clinical decision-makers to improve care quality and reduce the cost of inappropriate hospital and Accident and Emergency admissions. The aim of this study is to provide a review of modelling techniques and frameworks for predictive risk modelling of patients in hospital, based on routinely collected data such as the Hospital Episode Statistics database. A number of sub-problems can be considered such as Length-of-Stay and End-of-Life predictive modelling. The methodologies in the literature are mainly focused on addressing the problems using regression methods and Markov models, and the majority lack generalisability. In some cases, the robustness, accuracy and re-usability of predictive risk models have been shown to be improved using Machine Learning methods. Dynamic Bayesian Network techniques can represent complex correlations models and include small probabilities into the solution. The main focus of this study is to provide a review of major time-varying Dynamic Bayesian Network techniques with applications in healthcare predictive risk modelling.

Molecular characterization and phylogenetic analysis of a penaeidin-like antimicrobial peptide, Fi-penaeidin from Fenneropenaeus indicus

Antimicrobial peptides (AMPs) play a major role in innate immunity. Penaeidins are a family of AMPs that appear to be expressed in all penaeid shrimps. Penaeidins are composed of an N-terminal proline-rich domain, followed by a C-terminal domain containing six cysteine residues organized in two doublets. This study reports the first penaeidin AMP sequence, Fi-penaeidin (GenBank accession number HM243617) from the Indian white shrimp, Fenneropenaeus indicus. The full length cDNA consists of 186 base pairs encoding 61 amino acidswith an ORF of 42 amino acids and contains a putative signal peptide of 19 amino acids. Comparison of F. indicus penaeidin (Fi-penaeidin) with other known penaeidins showed that it shared maximum similarity with penaeidins of Farfantepenaeus paulensis and Farfantepenaeus subtilis (96% each). Fi-penaeidin has a predicted molecular weight (MW) of 4.478 kDa and theoretical isoelectric point (pI) of 5.3

Identificação e caracterização de um isolado de Hydrangea ringspot virus em hortênsia no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização de um isolado de Bidens mosaic virus proveniente de alface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.