Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a vitally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx wash also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response. (c) 2006 Elsevier Ltd. All rights reserved.

A Simple, Universal Colorimetric Assay for Endonuclease/Methyltransferase Activity and Inhibition Based on an Enzyme-Responsive Nanoparticle System

An enzyme responsive nanoparticle system that uses a DNA-gold nanoparticle (AuNP) assembly as the substrate has been developed for the simple, sensitive, and universal monitoring of restriction endonucleases in real time. This new assay takes advantage of the palindromic recognition sequence of the restriction nucleases and the unique optical properties of AuNPs and is simpler than the procedure previously described by by Xu et al. (Angew. Chem. Int. Ed. Engl. 2007, 46, 3468-3470). Because it involves only one type of ssDNA modified AuNPs, this assay can be directed toward most of the endonucleases by simply changing the recognition sequence found within the linker DNA. In addition, the endonuclease activity could be quantitatively analyzed by the value of the reciprocal of hydrolysis half time (t(1/2)(-1). Furthermore, our new design could also be applied to the assay of methyltransferase activity since the methylation of DNA inhibits its cleavage by the corresponding restriction endonuclease, and thus, this new methodology can be easily adapted to high-throughput screening of methyltransferase inhibitors.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32%similar to 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, A)Dnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana. (C) 2007 Published by Elsevier Inc.

毛白杨（Populus tomentosa）木质素生物合成相关基因分离与木质素合成调节

木质素是植物体中具重要生物功能的次生代谢产物。然而纸浆生产主要是将原料中的木质素与用于造纸的纤维素分离，该工艺过程产生了造纸工业的主要污染废液，并且增加造纸成本。本研究目的在于利用反义RNA技术，在分子水平调节木质素的生物合成，降低中国特有造纸树种毛白杨的木质素含量，培育更适于我国造纸工业的原料树种。以下为本研究已取得的相关研究进展： 1.通过RT-PCR技术，从毛白杨中克隆了木质素生物合成的三个相关酶的cDNAs，它们分别为咖啡酸甲基转移酶(caffeic acid O-methyltransferase，COMT)、咖啡酰CoA甲基转移酶(caffeoyl Co-enzyme A O-methyltransferase，CCoAOMT)及香豆酸：辅酶A连接酶(4-coumarate: CoA ligase，4CL)。序列分析显示了毛白杨这三个基因与杨属中其它种的相应基因cDNA核苷酸序列高度同源。Northern点杂交分析表明，COMT、CCoAOMT及4CL基因在毛白杨正在生长的次生木质部中高水平表达，其表达高峰与树木的木质化进程同步;而在叶与叶柄中，这三个基因均不表达。COMT、CCoAOMT及4CL是木质素生物合成的相关酶，该表达特征与其基因功能相一致。本研究克隆的COMT、CCoAOMT及4CL基因的cDNAs已在GenBank注册登记，接受号分别为AF237777、AF240466、AF314180 (publish on Jan l,2002)。 2.通过一系列的DNA重组，构建了携带反义COMT、CCoAOMT或4CLcDNA的反义表达载体以及同时整合反义COMT与CCoAOMT cDNA的双价反义表达载体，PCR扩增与酶切检测确证构建无误。 3.以田间取材的速生三倍体毛白杨B19、B331及B304的茎尖、叶片与嫩茎为外殖体，首次获得了三倍体毛白杨的组培再生试管苗，并建立了速生三倍体毛白杨的组培再生系统，为通过基因工程改良其造纸性能奠定了基础。 4.农杆菌介导转化烟草，PCR与PCR-Southern检测表明我们获得了整合反义COMT、CCoAOMT cDNA及反义COMT及CCoAOMT cDNA共整合的转基因烟草。以Digoxigenin标记的对应于反义链的单链RNA为探针与转基因烟草的总RNA进行NoIthern点杂交，结果表明整合到其中的反义cDNA均已表达。转基因烟草的木质素分析将有助于对COMT及CCoAOMT两个甲基化酶功能的认识。 5.通过农杆菌介导，将反义CCoAOMT cDNA转入欧洲山杨与银白杨的杂交杨（P tremulaXP.alba）。经PCR，PCR-Southern及Southern检测，确认获得了转基因植株。以Digoxigenin标记的对应于CCoAOMT cDNA反义链的单链RNA为探针与转基因杂交杨总RNA进行Northern点杂交，结果表明整合到其中的反义cDNA已在转录水平表达。测定生长5-6个月的转基因杨树下部茎杆的Klason木质素含量，结果显示其中一个株系的Klason木质素含量比野生型对照下降17.9%，表明抑制杨树内源CCoAOMT基因表达可有效降低转基因植株的木质素含量。

分子调控咖啡酸-O-甲基转移酶（COMT）对木质素合成及植物生长发育的影响

本论文由两部分组成，一、构建来自小麦的COMT的反义表达载体，转化烟草，研究抑制内源COMT对植物木质素合成及其生长发育的影响;二、利用花粉管通道法，将正义和反义COMT基因转化小麦，获得转基因小麦，从而进一步分析。 一、 反义抑制COMT对植物木质素合成及其生长发育的影响 构建含有小麦的咖啡酸-O-甲基转移酶（COMT）cDNA的反义表达载体, 利用农杆菌法转化烟草。 PCR, PCR-Southern 检测显示目的基因片段成功转入烟草基因组。处于营养生长期的转基因植株表型与对照没有明显差异;而发育成熟的转基因植株的植株矮化，茎部木质素含量与对照差异不大，木质素的组成S/G比下降，部分木质部细胞发生变形。我们还发现转基因烟草种子发芽率提高，移栽2个月的子一代转基因植株光合速率、蒸腾速率有所增强。结果表明通过反义抑制COMT将影响木质素合成,并在不同的发育阶段，影响着植物的生长发育。 二、 利用花粉管通道法获得转基因小麦 将构建好的含有Bar基因的正义和反义COMT表达载体利用花粉管法转化两个小麦品种（H4564和C6001），共获得转基因处理的种子1117颗，重新播种后，发育成苗分别为321株，总成苗率为28.7%。通过除草剂PPT筛选，分别获得PPT抗性植株31株。PCR检测抗性植株，获得PCR检测阳性植株5株，总阳性率为0.45%。阳性植株分别为H4564反义处理株1株，C6001的正义和反义处理株各2株。对小麦的植株高度，分蘖数等生理性状的分析发现，转基因小麦的分蘖数减少，植株高度降低。这些生理性状的改变与COMT基因转化的关系将有待于进一步验证。

喀斯特植物适应高钙的方式与干旱及外源钙诱导基因的克隆和表达

喀斯特环境是一类发育在可溶性碳酸盐类岩石上的环境系境，其生境具有高钙和干早等特征。喀斯特生境中的优势/特征植物也多具适应高钙、耐旱的特性。旋蒴苣苔(Boea hygrometrica)是喀斯特地区一种典型的抗旱和适应高钙的植物。本文在探讨了喀斯特地区植物适应高钙环境的不同方式的基础上，从旋蒴苣苔中克隆了两个受脱水和外源Ca2+诱导表达的基因，并对其表达调控和蛋白质产物进行了分析。本研究将为治理西南喀斯特地区日益退化的生态系统提供理论基础。 本文测定了贵州4个地区采集地内45种优势/常见种的地上部分和地下部分的全钙含量以及土壤的交换性钙含量。通过分析喀斯特地区植物与土壤钙含量的特征发现：喀斯特地区植物具有较高的钙含量平均值；土壤交换性钙含量对植物地上部钙含量的影响总体上不显著，对植物地下部钙含量的影响显著；不同类别的植物钙含量存在显著差异，蕨类植物地上部钙含量平均值明显低于被子植物；不同类别植物的钙的分布部位也存在显著差异，在蕨类植物和单子叶植物中地上部和地下部的钙含量相近，而双子叶植物的地上部钙含量明显高于地下部。根据地上部分与地下部分钙含量的差异性以及与土壤交换性钙含量的相关关系将其中14种优势植物对土壤高钙的适应方式分为3种类型：随遇型，高钙型和低钙型。随遇型植物地上部和地下部的钙含量均与土壤交换性钙含量成显著正相关关系；高钙型植物具有较强的钙富集能力，其地上部即使在低钙含量的土壤中也可维持较高的钙含量；低钙型植物的地上部即使在高钙的土壤中亦可维持较低的钙含量。对贵州和北京等地采集到的旋蒴苣苔钙含量和土壤含钙量的分析发现，其地上部和地下部钙含量即使在高钙土中也呈低水平，推测可能是通过控制根部对钙的吸收和向上运输来维持低钙水平。 从旋蒴苣苔cDNA 文库中筛选到两个片段，经5’RACE 得到cDNA 全长，分别命名为BhOMT1 和BhC2DP1。BhOMT1 编码一个包含359 个氨基酸的蛋白质，蛋白序列分析表明BhOMT1 为植物O-甲基转移酶。BhOMT1 基因组序列包含一个内含子，与其同源基因的基因结构相似。BhC2DP1 编码一个包含154 氨基酸的小分子蛋白质，内含一个C2 结构域，该结构域在很多植物蛋白中均存在。通过5’RAGE 的方法分别克隆了1465bp BhOMT1 启动子序列和1113bp 的BhC2DP1 启动子序列，分析发现其中均含有多种逆境胁迫诱导元件。通过对BhOMT1 和BhC2DP1 表达分析和Western 杂交发现这两个基因均受脱水和外源Ca2+的诱导表达，表明这两个基因可能参与了旋蒴苣苔的脱水保护和对高钙环境的适应。

Different protein tyrosine phosphatase superfamilies resulting from different gene reading frames

Protein tyrosine phosphatases (PTPs) are comprised of two superfamilies, the phosphatase I superfamily containing a single low-molecular-weight PTP (lmwPTP) family and the phosphatase II superfamily including both the higher-molecular-weight PTP (hmwPTP) and the dual-specificity phosphatase (DSP) families. The phosphatase I and H superfamilies are often considered to be the result of convergent evolution. The PTP sequence and structure analyses indicate that lmwPTPs, hmwPTPs, and DSPs share similar structures, functions, and a common signature motif, although they have low sequence identities and a different order of active sites in sequence or a circular permutation. The results of this work suggest that lmwPTPs and hmwPTPs/DSPs are remotely related in evolution. The earliest ancestral gene of PTPs could be from a short fragment containing about 90similar to120 nucleotides or 30similar to40 residues; however, a probable full PTP ancestral gene contained one transcript unit with two lmwPTP genes. All three PTP families may have resulted from a common ancestral gene by a series of duplications, fusions, and circular permutations. The circular permutation in PTPs is caused by a reading frame difference, which is similar to that in DNA methyltransferases. Nevertheless, the evolutionary mechanism of circular permutation in PTP genes seems to be more complicated than that in DNA methyltransferase genes. Both mechanisms in PTPs and DNA methyltransferases can be used to explain how some protein families and superfamilies came to be formed by circular permutations during molecular evolution.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Bromophenol derivatives from the red alga Rhodomela confervoides

Eight new bromophenol derivatives, 2,3-dibromo-4,5-dihydroxybenzyl methyl sulfoxide (1), 4-(2,3-dibromo-4,5-dihydroxyphenyl)-3-butene-2-one (2), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (3), 2-(3-bromo-5-hydroxy-4-methoxyphenyl)-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid methyl ester (4), 2-phenyl-3-(2,3-dibromo-4,5-dihydroxyphenyl)propionic acid (5), 4'-methoxy-2",3',3"-tribromo-4",5',5"-trihydroxydiphenylacetic acid (6), and 3-bromo-5-hydroxy-4-methoxyphenylacetic acid (7) and its methyl ester (8), together with a known bromophenol, 3-bromo-5-hydroxy4-methoxybenzoic acid (9), were isolated from the red alga Rhodomela confervoides. Their structures were elucidated by spectroscopic methods including IR, EIMS, FABMS, ESIMS, HRFABMS, HRESIMS, 1D and 2D NMR, and single-crystal X-ray structure analysis. Compounds 1-4, 8, and 9 were found inactive against several human cancer cell lines and microorganisms.