青蒿素生物合成调控的研究章焰生

青蒿 (Artemisia annua) 是目前唯一用来生产抗疟药剂青蒿素的一种药用植物，由于青蒿植株中青蒿素的含量过低，使得青蒿素的生产不能满足市场的需要，提高青蒿植株中青蒿素的含量显得尤为重要，本论文围绕着调控青蒿素的生物合成作出了以下一些研究： 一、紫穗槐二烯合酶基因启动子的克隆 　紫穗槐二烯合酶是参与青蒿素生物合成的一个关键酶，以法呢基焦磷酸为底物，催化形成青蒿素分子所具有的杜松烯环状结构, 此酶被认为是青蒿素分支途径中的第一个关键限速酶，因此它的表达水平高低关系着流向青蒿素生物合成的碳流量。为了了解紫穗槐二烯合酶的表达模式，用Tail-PCR的方法我们从青蒿高产株系001中克隆得到紫穗槐二烯合酶基因的两个启动子，分别命名为AMSP1与AMSP2，序列比较分析可看出两启动子有三处大的差异片段，由此推测紫穗槐二烯合酶基因在青蒿基因组中可能是一个多拷贝形式存在，Southern杂交验证了这一点，结果表明，紫穗槐二烯合酶基因在青蒿基因组中至少存在三个copy;利用PLACE数据库对克隆的启动子的顺式作用元件进行分析，发现其中有光、茉莉酸甲酯等调控元件。5'RACE分析确定了紫穗槐二烯合酶基因的转录起始位点位于翻译起始位点上游44bp处。通过PCR的方法，我们对启动子AMSP2进行5′端缺失，缺失的5个启动子片段克隆进报告基因GUS的上游，以进行下一步启动子的特性分析。 二、紫穗槐二烯合酶的表达特性分析 　　RT-PCR半定量分析表明：光正调控于紫穗槐二烯合酶的转录表达，黑暗培养条件下，青蒿植株中紫穗槐二烯合酶的转录水平较低，一旦进行光照培养，紫穗槐二烯合酶的转录水平显著上升;茉莉酸甲酯对青蒿植株中紫穗槐二烯合酶的表达调控具有光依赖性，在连续黑暗条件下，茉莉酸甲酯对紫穗槐二烯合酶的转录水平并无影响，而在正常光照与黑暗循环培养条件下，茉莉酸甲酯明显诱导了青蒿植株中紫穗槐二烯合酶的转录表达。鉴于在正常的培养条件下，茉莉酸甲酯正调控于紫穗槐二烯合酶的转录表达，我们研究了茉莉酸甲酯对青蒿植株中青蒿素生物合成的影响，结果表明，茉莉酸甲酯并未促进青蒿素的生物合成，却诱导了青蒿素生物合成的前体青蒿酸的生物合成。 三、真空介导农杆菌转化青蒿植株瞬时表达系统的建立 　　探讨了真空强度、青蒿苗龄、农杆菌的浓度、共培养方式、共培养时间以及光对青蒿植株瞬时表达的影响，结果表明真空强度70－80mbar、30天的青蒿苗龄以及侵染农杆菌浓度OD600为2.0时有利于青蒿植株的瞬时表达;液体共培养极大地抑制了外源基因在青蒿植株的瞬时表达，固体共培养与滤纸共培养适合于青蒿植株的瞬时表达系统，二者之间无明显差异;共培养时间也是影响基因瞬时表达的关键因素，结果表明，3天或4天较2天共培养有利于外源基因在青蒿植株的瞬时表达;光对青蒿植株的瞬时表达有极大地抑制作用，而黑暗条件适合于青蒿植株的瞬时表达;此系统可用于不同基因型的青蒿植株。 四、青蒿过氧化物酶基因的克隆及功能分析 　　利用RACE方法，从青蒿高产株系001中克隆了一个第三大类植物过氧化物酶的cDNA。克隆的青蒿过氧化物酶氨基酸序列与白羽扇豆、辣根菜、小麦、烟草、蕃茄的过氧化物酶的同源性分别为42.0%、36.2%、38.9%、33.6%和32.8%。青蒿过氧化物酶的编码区被克隆进原核表达载体PET-30a，并在大肠杆菌BL21(DE3)pLysS中诱导表达，过量表达产物主要以包涵体形式存在，但也有相当一部分可溶性蛋白出现。表达的蛋白具有明显催化抗坏血酸、愈创木酚的过氧化物酶活性，催化愈创木酚活性大约是催化抗坏血酸的1.8倍。氨基酸同源性分析与过氧化反应表明克隆的过氧化物酶属于植物第三大类过氧化物酶。青蒿过氧化物酶间接促进了青蒿细胞提取液中青蒿酸向青蒿素的生物转化，但不能直接以青蒿酸作为催化底物。 五、外源赤霉素处理与开花对青蒿素、青蒿酸生物合成的影响 　　研究结果发现在青蒿的营养生长期喷施外源赤霉素明显促进了青蒿素的生物合成，同时青蒿酸含量呈下降趋势;从营养期至生殖期，青蒿酸的含量逐渐下降，至开化前期青蒿酸含量下降到最低，从开化前期至开花后期，青蒿酸的含量无多大变化，随着青蒿的发育，从营养生长期至开花期，青蒿素的含量呈上升趋势，至开化盛期时青蒿素含量达到最高。外源赤霉素处理与开花打破了青蒿素生物合成的瓶颈，诱导了青蒿酸向青蒿素的生物转化。

复苏植物牛耳草Boea hygrometrica耐旱复苏过程中LEA基因表达调控及功能研究

本实验室以复苏被子植物旋蒴苣苔（Boea hygrometrica (Bunge) R Br，牛耳草）为实验材料，利用cDNA微阵列技术，筛选到2个LEA蛋白的cDNA片段。它们在干旱条件下表达水平增加一倍以上。在上述基础上，通过5’-RACE的方法扩增得到这两个LEA基因的全长cDNA，BhLEA1和BhLEA2，发现二者编码蛋白均与玄参科复苏植物Craterostigma plantagineum中的第4组LEA蛋白pcCC2具有最高的同源性。PCR扩增得到的基因组序列中发现BhLEA1和BhLEA2在靠近5’端处各有一内含子，分别为97和171bp。利用tail-PCR和inverse-PCR扩增得到BhLEA2的1215bp的启动子序列，其中含ABRE、W-box、HDEs、MYB、MYC、和生长素响应元件等。两个基因在不同胁迫、激素等处理下都有诱导表达。构建过量表达载体, 转化烟草。筛选T2代的纯合体植株进行抗旱试验，测定了干旱过程中的土壤和叶片含水量，PSⅡ最大光化学效率Fv/Fm、SOD和POD酶的活性及蛋白质降解程度，发现BhLEA1和BhLEA2过量表达的烟草抗旱性有明显提高，与野生型相比，叶片含水量和光系统Ⅱ活性下降慢、SOD和POD活性增高、蛋白质降解减少。Western blot检测发现在干旱时，野生型烟草的RbcL、LHCⅡ和PsBO蛋白明显降解，而转基因植物的几乎没有降解，或者降解较少，说明BhLEA的过量表达可以在一定程度上抑制干旱诱导的蛋白质降解。这些结果表明，BhLEA基因可能直接或间接地在牛耳草干旱过程中参与了保护蛋白质的作用，为牛耳草叶片的复苏提供了基础。

LPA3蛋白参与调控PSII组装的功能研究

PSII是一个叶绿体类囊体膜的蛋白复合体，它由20多个蛋白亚基组成，这些蛋白由核基因和叶绿体基因共同编码。由于PSII结构的复杂性，PSII的组装是多步骤的，并得到辅因子和调控蛋白的协助。但我们对参与调控步骤的蛋白因子还了解不多。鉴于叶绿体有限的编码能力，推测参与叶绿体组装调控的因子主要是由核基因来编码的。辨定这些核编码的叶绿体蛋白并深入研究其作用的分子机制将有助于我们了解PSII生物发生的分子机理。为此，我们利用叶绿素荧光成像系统对pER16 T-DNA插入突变体库进行了筛选，并对高荧光突变体lpa3进行了研究。主要研究结果如下： 突变体lpa3生长较为缓慢，叶色黄绿，叶绿素含量低。在突变体中，最大荧光量子产率Fv/Fm降低到0.514，表明PSII光合功能受到了损伤。突变体lpa3叶绿素荧光慢诱导曲线的正常下降表明PSII后的电子传递正常。突变体P700氧化还原动力学与野生型一致，则进一步表明PSI在突变体中是具有功能的。77K发射荧光光谱显示PSII的特征峰在突变体中较高，而PSI的特征荧光峰没有变化，则进一步显示突变体lpa3是一个PSII突变体。 通过Tail-PCR，发现突变体中T-DNA插入导致基因lpa3缺失表达，并且lpa3基因的互补可以使突变体的性状得到恢复。该基因表达的蛋白LPA3含有一个跨膜区域，是一个类囊体膜蛋白。该蛋白不是PSII的蛋白组成成分，可能与自身或者其它的蛋白组成一个复合体而起作用。 在突变体lpa3中PSII蛋白质尤其是核心蛋白D1、D2，含量下降。并且突变体中PSII蛋白复合体含量下降。但是突变体中PSII基因的表达并没有在转录水平受到调节，并且蛋白D1和D2的翻译起始也没有受到影响。体外标记实验表明，PSII中D1蛋白的合成明显降低，而其它蛋白的合成没有改变。进一步实验表明，突变体中PSII的组装效率降低。推测D1蛋白由于不能有效组装而反馈调节自身的合成。 酵母双杂交实验表明LPA3蛋白可以与D1蛋白相互作用，并且也与参与PSII组装的蛋白Alb3相互作用。因此，LPA3蛋白可能和蛋白Alb3形成一个复合体，该复合体与D1蛋白直接相互作用而参与调控PSII的组装。

Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization

The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution. (C) 2008 Published by Elsevier Inc.

Cloning and characterization of the gene for UDPGlc dehydrogenase from the cyanobacterium, Microcystis aeruginosa FACHB 905

Using degenerate primers based on conserved regions of the UDP-glucose dehydrogenase (UDPGDH) gene, an initial 476-bp DNA fragment was amplified from the water-bloom forming cyanobacterium, Microcystis aeruginosa FACHB 905. TAIL-PCR and ligation-mediated PCR were used to amplify the flanking regions to isolate an about 2.5-kb genomic DNA fragment. Sequence analysis revealed an ORF encoding a putative 462 amino acid protein, designated Mud for Microcystis UDPGDH. The Mud amino acid sequence is closely related to UDPGDH sequences from cyanobacterium Synechocystis PCC6803 (73% identity, 81% similarity), and bacterium Bacillus subtilis (51% identity and 67% similarity). The cloned mud gene was expressed in Escherichia coli using the pGEX-4T-1 fusion expression vector system to generate a GST-Mud fusion protein that exhibited UDPGDH activity. The cytosolic fraction of M aeruginosa FACHB 905 was subjected to Western analysis with an anti-Mud antibody, which revealed a single band of approximately 49 kD, consistent with the deduced molecular mass of the enzyme. The Mud protein could thus be characterized as a UDP-glucose dehydrogenase, which was a key enzyme for polysaccharide synthesis and has, for the first time, been studied in algae.

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.

β-glucosidases from Aspergillus unguis NII 08123: Molecular characterization, properties and applications

Lignocellulosic biomass is probably the best alternative resource for biofuel production and it is composed mainly of cellulose, hemicelluloses and lignin. Cellulose is the most abundant among the three and conversion of cellulose to glucose is catalyzed by the enzyme cellulase. Cellulases are groups of enzymes act synergistically upon cellulose to produce glucose and comprise of endoglucanase, cellobiohydrolase and β-glucosidase. β -glucosidase assumes great importance due to the fact that it is the rate limiting enzyme. Endoglucanases (EG) produces nicks in the cellulose polymer exposing reducing and non reducing ends, cellobiohydrolases (CBH) acts upon the reducing or non reducing ends to liberate cellobiose units, and β - glucosidases (BGL) cleaves the cellobiose to liberate glucose completing the hydrolysis. . β -glucosidases undergo feedback inhibition by their own product- β glucose, and cellobiose which is their substrate. Few filamentous fungi produce glucose tolerant β - glucosidases which can overcome this inhibition by tolerating the product concentration to a particular threshold. The present study had targeted a filamentous fungus producing glucose tolerant β - glucosidase which was identified by morphological as well as molecular method. The fungus showed 99% similarity to Aspergillus unguis strain which comes under the Aspergillus nidulans group where most of the glucose tolerant β -glucosidase belongs. The culture was designated the strain number NII 08123 and was deposited in the NII culture collection at CSIR-NIIST. β -glucosidase multiplicity is a common occurrence in fungal world and in A.unguis this was demonstrated using zymogram analysis. A total 5 extracellular isoforms were detected in fungus and the expression levels of these five isoforms varied based on the carbon source available in the medium. Three of these 5 isoforms were expressed in higher levels as identified by the increased fluorescence (due to larger amounts of MUG breakdown by enzyme action) and was speculated to contribute significantly to the total _- β glucosidase activity. These isoforms were named as BGL 1, BGL3 and BGL 5. Among the three, BGL5 was demonstrated to be the glucose tolerant β -glucosidase and this was a low molecular weight protein. Major fraction was a high molecular weight protein but with lesser tolerance to glucose. BGL 3 was between the two in both activity and glucose tolerance.121 Glucose tolerant .β -glucosidase was purified and characterized and kinetic analysis showed that the glucose inhibition constant (Ki) of the protein is 800mM and Km and Vmax of the enzyme was found to be 4.854 mM and 2.946 mol min-1mg protein-1respectively. The optimumtemperature was 60°C and pH 6.0. The molecular weight of the purified protein was ~10kDa in both SDS as well as Native PAGE indicating that the glucose tolerant BGL is a monomeric protein.The major β -glucosidase, BGL1 had a pH and temperature optima of 5.0 and 60 °C respectively. The apparent molecular weight of the Native protein is 240kDa. The Vmax and Km was 78.8 mol min-1mg protein-1 and 0.326mM respectively. Degenerate primers were designed for glycosyl hydrolase families 1, 3 and 5 and the BGL genes were amplified from genomic DNA of Aspergillus unguis. The sequence analyses performed on the amplicons results confirmed the presence of all the three genes. Amplicon with a size of ~500bp was sequenced and which matched to a GH1 –BGL from Aspergillus oryzae. GH3 degenerate primers producing amplicons were sequenced and the sequences matched to β - glucosidase of GH3 family from Aspergillus nidulans and Aspergillus acculateus. GH5 degenerate primers also gave amplification and sequencing results indicated the presence of GH5 family BGL gene in the Aspergillus unguis genomic DNA.From the partial gene sequencing results, specific as well as degenerate primers were designed for TAIL PCR. Sequencing results of the 1.0 Kb amplicon matched Aspergillus nidulans β -glucosidase gene which belongs to the GH1 family. The sequence mainly covered the N-Terminal region of the matching peptide. All the three BGL proteins ie. BGL1, BGL3 and BGL5 were purified by chromatography an electro elution from Native PAGE gels and were subjected to MALDI-TOF mass spectrometric analysis. The results showed that BGL1 peptide mass matched to . β -glucosidase-I of Aspergillus flavus which is a 92kDa protein with 69% protein coverage. The glucose tolerant β -glucosidase BGL5 mass matched to the catalytic C-terminal domain of β -glucosidase-F from Emericella nidulans, but the protein coverage was very low compared to the size of the Emericella nidulans protein. While comparing the size of BGL5 from Aspergillus unguis, the protein sequence coverage is more than 80%. BGL F is a glycosyl hydrolase family 3 protein.The properties of BGL5 seem to be very unique, in that it is a GH3 β -glucosidase with a very low molecular weight of ~10kDa and at the same time having catalytic activity and glucose 122 tolerance which is as yet un-described in GH β -glucosidases. The occurrence of a fully functional 10kDA protein with glucose tolerant BGL activity has tremendous implications both from the points of understanding the structure function relationships as well as for applications of BGL enzymes. BGL-3 showed similarity to BGL1 of Aspergillus aculateus which was another GH3 β -glucosidase. It may be noted that though PCR could detect GH1, GH3 and GH5 β-glucosidases in the fungus, the major isoforms BGL1 BGL3 and BGL5 were all GH3 family enzymes. This would imply that β-glucosidases belonging to other families may also co-exist in the fungus and the other minor isoforms detected in zymograms may account for them. In biomass hydrolysis, GT-BGL containing BGL enzyme was supplemented to cellulase and the performances of blends were compared with a cocktail where commercial β- glucosidase was supplemented to the biomass hydrolyzing enzyme preparation. The cocktail supplemented with A unguis BGL preparation yielded 555mg/g sugar in 12h compared to the commercial enzyme preparation which gave only 333mg/g in the same period and the maximum sugar yield of 858 mg/g was attained in 36h by the cocktail containing A. unguis BGL. While the commercial enzyme achieved almost similar sugar yield in 24h, there was rapid drop in sugar concentration after that, indicating probably the conversion of glucose back to di-or oligosaccharides by the transglycosylation activity of the BGl in that preparation. Compared this, the A.unguis enzyme containing preparation supported peak yields for longer duration (upto 48h) which is important for biomass conversion to other products since the hydrolysate has to undergo certain unit operations before it goes into the next stage ie – fermentation in any bioprocesses for production of either fuels or chemicals.. Most importantly the Aspergillus unguis BGL preparation yields approximately 1.6 fold increase in the sugar release compared to the commercial BGL within 12h of time interval and 2.25 fold increase in the sugar release compared to the control ie. Cellulase without BGL supplementation. The current study therefore leads to the identification of a potent new isolate producing glucose tolerant β - glucosidase. The organism identified as Aspergillus unguis comes under the Aspergillus nidulans group where most of the GT-BGL producers belong and the detailed studies showed that the glucose tolerant β -glucosidase was a very low molecular weight protein which probably belongs to the glycosyl hydrolase family 3. Inhibition kinetic studies helped to understand the Ki and it is the second highest among the nidulans group of Aspergilli. This has promoted us for a detailed study regarding the mechanism of glucose tolerance. The proteomic 123 analyses clearly indicate the presence of GH3 catalytic domain in the protein. Since the size of the protein is very low and still its active and showed glucose tolerance it is speculated that this could be an entirely new protein or the modification of the existing β -glucosidase with only the catalytic domain present in it. Hydrolysis experiments also qualify this BGL, a suitable candidate for the enzyme cocktail development for biomass hydrolysis

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

alpha(1)-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats

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

Differentiation of Haemonchus placei from Haemonchus contortus by PCR and by morphometrics of adult parasites and third stage larvae

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

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic E. coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.