白氏文昌鱼FADD的克隆及功能研究

Fas死亡结构域相关蛋白(Fas-associated death domain protein,FADD)是死亡信号转导通路中的连接蛋白,在脊椎动物中其结构和功能都很保守。本文首次克隆了头索动物白氏文昌鱼(Branchiostoma belcheri)FADD(bbFADD)的cDNA和基因组DNA序列。bbFADDcDNA全长1239 bp,编码217个氨基酸。与脊椎动物的FADD一样,bbFADD含有N端的死亡效应结构域(Death Effector Domain,DED)和C端的死亡结构域(Dea

Enhanced grass carp reovirus resistance of Mx-transgenic rare minnow (Gobiocypris rarus)

In the interferon-induced antiviral mechanisms, the Mx pathway is one of the most powerful. Mx proteins have direct antiviral activity and inhibit a wide range of viruses by blocking an early stage of the viral genome replication cycle. However, antiviral activity of piscine Mx remains unclear in vivo. In the present study, an Mx-like gene was cloned, characterized and gene-transferred in rare minnow Gobiocypris rarus, and its antiviral activity was confirmed in vivo. The full length of the rare minnow Mx-like cDNA is 2241 bp in length and encodes a polypeptide of 625 amino acids with an estimated molecular mass of 70.928 kDa and a predicted isoelectric point of 7.33. Analysis of the deduced amino acid sequence indicated that the mature peptide contains an amino-terminal tripartite GTP-binding motif, a dynamin family signature sequence, a GTPase effector domain and two carboxy-terminal leucine zipper motifs, and is the most similar to the crucian carp (Carassius auratus) Mx3 sequence with an identity of 89%. Both P0 and F1 generations of Mx-transgenic rare minnow demonstrated very significantly high survival rate to GCRV infection (P < 0.01). The mRNA expression of Mx gene was consistent with survival rate in F1 generation. The virus yield was also concurrent with survival time using electron microscope technology. Rare minnow has Mx gene(s) of its own but introducing more Mx gene improves their resistance to GCRV. Mx-transgenic rare minnow might contribute to control the GCRV diseases. (C) 2008 Published by Elsevier Ltd.

Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

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.

天然免疫相关分子的进化

本论文结合功能研究和进化遗传学方法对动物天然免疫（innate immunity）相关分子的进化历程进行深入研究。受体对病原微生物的识别是天然免疫系统发挥功能的基础。作为模式识别受体（pattern recognition receptor, PRR），果蝇肽聚糖识别蛋白SD（PGRP-SD）在识别革兰氏阳性细菌的过程中发挥了重要作用。针对已有的黑腹果蝇（Drosophila melanogaster）群体数据，我们发现PGRP-SD在群体中存在2类高频的等位基因（分别为等位基因1和等位基因2）。以D. simulans为外群，我们追溯了黑腹果蝇2类等位基因上氨基酸的变化。这些氨基酸的结构特征和在蛋白质上所处的位置提示这2类等位基因在功能方面可能存在分化。通过功能研究的方法，我们发现在黑腹果蝇中该基因功能方面发生了显著的变化。等位基因2在有微生物时能激活天然免疫反应，但等位基因1的转基因果蝇成虫只要有外伤即便没有微生物的情况下即能激发天然免疫反应，而带有等位基因2果蝇成虫则不具有该功能。这一结果提示我们，发生在该等位基因上的氨基酸变化导致了其识别功能的变化。与推导的祖先基因相比，等位基因1发生了一个氨基酸的变化，因此导致其功能从识别细菌细胞壁组分肽聚糖转变为一未知的自身组分，即从病原相关分子模式（pathogen-associated molecular pattern，PAMP）识别受体转变为损伤相关识别模式（damage-associated molecular pattern, DAMP）识别受体。通过这一功能变化， 果蝇成虫可以通过仅识别自身损伤即可激活相应的免疫反应，对后续可能侵入的微生物进行杀伤。已有研究结果显示，微生物在进化过程中已经形成针对DAMP和PAMP规避策略。上述2类等位基因的同时存在能使黑腹果蝇同时具备两个机制，更加充分地抵抗病原微生物的入侵。结合功能研究和针对自然群体的群体遗传学分析，我们认为在黑腹果蝇群体中以高频共存的2类PGRP-SD等位基因可能可能受到了平衡选择（balancing selection）作用。上述工作主要研究了天然免疫系统识别受体的进化。而本论文的另一部分则主要针对天然免疫系统的效应分子（effector）进行了研究。作为重要的效应分子，抗菌肽在杀菌方面发挥着最为直接的作用。因此，研究抗菌肽的进化对于探索天然免疫系统的进化具有重要意义。本研究以两栖类动物大蹼铃蟾抗菌肽基因家族为例，通过对分别来自2个大蹼铃蟾个体的皮肤cDNA文库进行测序，我们鉴别出56个不同的抗菌肽cDNA序列。每一个cDNA均编码2个不同的抗菌肽，maximin 和maximin H。基于针对这些cDNA序列的分析，我们发现2类抗菌肽编码序列的非同义替代率均高于同义替代率，呈现高度分化的特征。但是，在信号肽和其它非抗菌肽编码区域并没有发现这种情况。这一结果提示抗菌肽可能受到超显性选择（overdominent selection, 即平衡选择）的影响。同时，我们分别从皮肤和肝脏克隆基因了7个抗菌肽的基因组编码序列并进行了测序。这些从不同组织获得的抗菌肽在各个编码序列中均存在序列的差异的同时呈现了相同的结构。这一结果提示不同抗菌肽间的差异不太可能来自于体细胞突变而是快速序列进化的结果。通过构建来自于同一个体的抗菌肽的不同编码区的基因树，我们发现结构域重排（domain shuffling）和/或基因转换（gene conversion）在这些抗菌肽的进化历程中发挥作用。

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

一种离散驱动蛇形柔性臂的工作空间及优化设计研究

以一种离散驱动蛇形柔性臂为研究对象 ,分析并提出了弯曲型离散驱动机器人工作空间的表达方法。考虑到离散驱动机器人工作空间的离散性 ,为了便于对其运动学参数进行调整 ,以使末端操作器达到新指定的工作位置 ,提出了参数调整“标定”的概念 ,并依据使运动学参数调整值尽量小的优化原则对运动学参数进行了优化设计 ,从而解决了运动学参数设计的冗余度问题。

并联Stewart机构位姿误差分析

从并联机构与串联机构的运动学等效 ,并联机构本身特征与并联机构实际工作空间出发 ,考虑各分支末端误差对最终运动平台末端误差的影响 ,提出了并联机构位姿误差放大因子分析法·依据位姿误差放大因子具有对误差定量分析的特点 ,该分析方法既可用于机构参数优化 ,又可用于结构精度设计· 最后 ,给出了一个实例说明本方法的有效性·

主从机械手遥控操作摄像机自动跟踪系统

介绍了一种摄像机云台自动跟踪控制系统 ,该系统用来实现遥控机械手主从操作时摄像机自动跟踪从手末端夹钳的运动以辅助操作员进行遥控操作 ;介绍了系统的组成、工作原理及控制算法 ;实验结果验证了算法的正确性和控制系统的可行性。

PUMA760切割作业力与位置混合控制

本文介绍在 PUMA760 上实现了的切割作业控制系统。我们在 PUMA760 控制系统中引进了力传感器信号,加入了力控制算法,通过调整机器人手部位置,控制机器人与环境物体间的相互作用力。在 VAL 系统中加入了根据传感器信号动作的指令,实现了一种机器人力与位置的混合控制。用扩充后的 VAL 系统编程,成功地使 PUMA760完成了多种材料的切割作业。

用光学反射传感器的机器人初始定位系统

本文叙述一种用光学反射传感器自动确定机器人在工作坐标中的起始参考位置的方法。装在机器人手爪边上的光学反射传感器与工作面上的T形目标相配合,用自动反复循环定位法确定机器人在工作坐标中的期望起始点。实践和理论分析表明,虽然开机时的机器人手爪的原始位置基本上是随机的,但手爪都能自动精确地收敛到期望的起始点上。