细胞分裂素的测定及其转基因植物的基因表达和调控的研究

细胞分裂素是一类重要的植物激素，它参与调节许多植物的生命活动过程。本文从几个方面研究了细胞分裂素的作用。 在细胞分裂素的活性测定中，通过改进尾穗蔸苋红素合成法建立了一种简便、准确的生物试法，同时还建立了根据物理化学和免疫学原理而测定细胞分裂素的HPLC和ELISA方法，使得细胞分裂素的定量更加准确。经过对上述三种方法的相互验证实验表明，同时采用二种方法可以保证细胞分裂素分析的准确和可靠。 细胞分裂素可以促进黄瓜子叶的扩张。利用离体黄瓜子叶，分析BA诱发其扩张与子叶内源细胞分裂素之间的关系，实验证明，BA能促进玉米素及其核苷的迅速积累，进而诱发子叶的扩张。上述结果还表明，黄瓜子叶可能具有合成细胞分裂素的能力。 荸荠球茎是一种贮藏器官，但实验测定发现其中含有细胞分裂素的生理活性形式——异戊烯基腺嘌呤核苷(iPA)，而且合成它的前体腺嘌呤的含量也十分丰富，考虑到球茎与种子的类似之处，推测它可能做为合成细胞分裂素的一个源，而且其合成途径可能有别于植物其它组织。 农杆菌中的异戊烯基转移酶(ipt)基因是负责细胞分裂素生物合成的关键基因。将ipt基因克隆后对其启动子进行了改造，分别构建了如下三种基因：(1) ipt启动子+ipt编码区和3，区(ipt)，(2)磷酸核酮糖羧化酶小亚基启动子SSU 301+ipt编码区和3，区(SSU -ipt)，(3)豌豆种子特异性启动子viciln+ipt编码区和3，区(vic-ipt)。上述三种基因经农杆菌介导转化烟草，获得了16株再生植株，经Southern杂交证明其中15株的基因组上含有正常整合的ipt基因。Northern杂交表明有13株转基因烟草中的ipt基因能转录出大小正常的ipt mRNA并促进了细胞分裂素的生物合成。 实验表明，转基因烟草中ipt基因的表达受到多种因素的调控。首先启动子决定了ipt基因的表达模式，SSU -ipt基因的表达受光的诱导，黑暗中这种基因的转录完全停止，而vic-ipt基因的表达是种子特异性的，它不在烟草营养生长器官如根、茎、叶和愈伤组织中表达。第二，生长素能降低ipt基因的表达活性。第三，在整体植物的根中，存在某些反式因子，能够控制ipt基因的过量表达，这其中可能涉及到细胞内的蛋白因子、基因的甲基化作用及细胞分裂素的反馈调节等。 vic-ipt基因在烟草种子中的特异性表达导致种子内形成了一个细胞分裂素合成的源(source)。对种子中营养物质积累的研究表明，ipt基因的表达促进了种子干物质的积累，其中作用最明显的是增加种子内蛋白质的合成。转入vic-ipt基因后的烟草种子其萌发率没有显著变化，但幼苗的生长速率明显加快，这表明细胞分裂素能调节植株的生长。 通过Northern杂交检测转基因烟草中基因表达的调控，实验证明，ipt基因的表达明显抑制一组植物病理相关蛋白(PR)基因的转录活性，这组基因编码：几丁质酶，β-1，3一葡萄糖苷酶，伸展蛋白和渗调蛋白。对这些调控作用的生理学意义还有待进一步探索。 上述结果表明，在高等植物中，除了传统上认为根是合成细胞分裂素的部位之外，其它组织和器官也具有合成细胞分裂素的能力，其中合成能力最强的是一些离体组织和贮藏器官。农杆菌中的细胞分裂素生物合成基因(ipt)能够在高等植物的基因组中正常的整合和表达，并受到植物体内生理、发育等多种因素的调控，而与整体植物的正常生理过程协调一致。ipt基因的表达还能够调节植物体的生长和发育，包括种子发育时营养物质的积累、幼苗的生长和某些相关基因的表达。对上述问题的深入研究,必将促进细胞分裂素及其相关生理学和发育学研究的进展。

重金属胁迫相关基因PvSR2在大肠杆菌中表达和转化烟草

近年来植物重金属的耐性机制研究及抗重金属基因工程取得了很大进展。本文将来自于菜豆(Phaseolus vulgaris)的特异性重金属胁迫相关基因PvSR2 (Phaseolus vulgaris stress-related protein, PvSR)的cDNA序列克隆到大肠杆菌高效表达载体pBV221的PR PL启动子的下游，构建了原核表达载体pBV221-PvSR2。通过温度诱导，在大肠杆菌中成功地高效表达了PvSR2基因。经重金属(CdCl2)抗性检测，实验组比对照组有明显的抗性。 同时，将该基因克隆到植物转达化中间载体pCAMBEIA2301的花椰菜花椰病毒的35S启动子下游，利用根癌农杆菌(Agrobacterium tumefaciens)Ti质粒介导的遗传转化系统，成功地将该基因导入了烟草的基因组，获得了转基因植株。

大蹼铃蟾皮肤膜联蛋白Ⅱ相关蛋白的分离纯化及活性

通过阴离子交换、凝胶过滤和阳离子交换层析, 从大蹼铃蟾皮肤中纯化到一个表观分子量为33 kDa 的单链蛋白。N - 末端序列比较分析显示, 该蛋白与来自非洲爪蟾、红色原鸡和人膜联蛋白Ⅱ的N - 末端序列 相同的氨基酸分别占70 %、64 %和56 %。该蛋白具有以钙依赖的方式抑制专一性血小板膜糖蛋白Ⅵ受体激动剂 ———Stejnulxin 诱导洗涤人血小板聚集的生物学功能, 最大抑制率达48 %。结合其N - 末端序列BLAST 搜索结 果及其活性的钙依赖性, 推测该蛋白是与膜联蛋白Ⅱ相关的一类蛋白质。

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors - a commentary on the spirochaete origin of flagella

The origin of cytoskeleton and the origin of relevant intracellular transportation system are big problems for understanding the emergence of eukaryotic cells. The present article summarized relevant information of evidences and molecular traces on the origin of actin, tubulin, the chaperonin system for folding them, myosins, kinesins, axonemal dyneins and cytoplasmic dyneins. On this basis the authors proposed a series of works, which should be done in the future, and indicated the ways for reaching the targets. These targets are mainly: 1) the reconstruction of evolutionary path from MreB protein of archaeal ancestor of eukaryotic cells to typical actin; 2) the finding of the MreB or MreB-related proteins in crenarchaea and using them to examine J. A. Lake's hypothesis on the origin of eukaryote from "eocytes" (crenarchaea); 3) the examinations of the existence and distribution of cytoskeleton made of MreB-related protein within coccoid archaea, especially in amoeboid archaeon Thermoplasm acidophilum; 4) using Thermoplasma as a model of archaeal ancestor of eukaryotic cells; 5) the searching for the homolog of ancestral dynein in present-day living archaea. During the writing of this article, Margulis' famous spirochaete hypothesis on the origin of flagella and cilia was unexpectedly involved and analyzed from aspects of tubulins, dyneins and spirochaetes. Actually, spirochaete cannot be reasonably assumed as the ectosymbiotic ancestor of eukaryotic flagella and cilia, since their swing depends upon large amount of bacterial flagella beneath the flexible outer wall, but not depends upon their intracellular tubules and the assumed dyneins. In this case, if they had "evolved" into cilia and lost their bacterial flagella, they would immediately become immobile! In fact, tubulin and dynein-like proteins have not been found in any spirochaete.

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 mug/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HC1 buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin a(11b)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca2+ with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca2+ release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLC gamma 2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity. (c) 2005 Elsevier Inc. All rights reserved.

Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor

A highly active cobra venom factor (CVF) was isolated from the venom of Naja kaouthia by sequential column chromatography. It displays strong anticomplementary activity, and has 1515 U of anti complementary activity per mg protein. A single dose of 0.1 mg/kg CVF given i.v. to rats completely abrogated complement activity for nearly 5 days. Given 0.02 mg/kg of CVF. the complement activity of rats was reduced by more than 96.5% in 6 It. In guinea pig-to-rat heart transplant model, rats treated with a single dose of 0.05 mg/kg CVF had significantly prolonged xenograft survival (56.12 +/- 6.27 h in CVF-treated rats vs. 0.19 +/- 0.07 h in control rats, P < 0.001). (C) 2003 Elsevier Ltd. All rights reserved.

Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

Effects of cyanobacterial toxin microcystin-LR on the transcription levels of immune-related genes in grass carp Ctenopharyngodon idella

Recent studies in mammals have revealed that the cyanobacterial toxin MC-LR suppresses immune functions. Nevertheless, immunotoxic effects of microcystins have been little studied in fish. In this paper, we present the profiles of the immune modulation of MC-LR in grass carp, and quantitative real-time PCR methodology was developed for the measurement of relative transcription changes of six immune-related genes in the spleen and head kidney of the grass carp Ctenopharyngodon idella, which were intraperitoneally injected with 50 mu g MC-LR center dot kg(-1) body weight in a three-week period. This study was focused exclusively on gene transcription level changes at different time points after MC-LR exposure, so, only one dose was given. The investigated genes were interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), type I interferon (Type I IFN), peptidoglycan recognition protein-L (PGRP-L), immunoglobulin M (IgM) and major histocompatibility complex class I (MHC-I) genes. The results demonstrated that the transcription levels of the TNF-alpha, type I IFN, and PGRP-L genes in the spleen and head kidney were significantly low at all time points, and those of IL-1 beta were significantly low in the head kidney at different time points. In addition, IgM and MHC-I transcription levels were only significantly low in the spleen and head kidney at 21 d postinjection. The changes in the transcription levels of immune-related genes induced by MC-LR confirmed its effect on inhibiting immune function at the transcription level.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

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.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.