具有III型分泌系统的Pseudomonas moraviensis FP1761植物益生功能及其全基因组分析

 本研究从节瓜根际分离到一株具有防病促生功能的荧光类假单胞菌FP1761。该菌株对部分植物病原真菌和细菌具有拮抗能力,能够解钾、解有机磷和无机磷,可产生氨、蛋白酶、嗜铁素、吲哚乙酸。生物测定表明菌株FP1761可显著促进小麦生长。生理生化、平均核苷酸相似度、16S rDNA和多基因分析将FP1761鉴定为摩拉维亚假单胞菌(Pseudomonas moraviensis)。菌株FP1761基因组草图全长6.12 Mb,(G+C)含量为59.9%,共编码5467个基因序列。将该菌株与种内3个代表性菌株进行泛基因组和核心基因组分析,共产生 4 357个共有基因,菌株FP1761特有基因327个。利用antiSMASH对菌株次生代谢基因簇进行预测,发现其含有8个潜在的次生代谢产物基因簇。其中两个基因簇与嗜铁素pyoverdine合成相关,未见聚酮类合成基因。基因组分析发现,该菌株具有与病原性假单胞菌相似的III型分泌系统,但丢失了效应蛋白调控因子hrpS和转运相关的hrpH和hrpK1基因。对全基因组扫描,菌株FP1761仅保留了病原性假单胞菌的保守效应蛋白AvrE和HopAA1-1。FP1761是目前已发现的唯一具有III型分泌系统的摩拉维亚假单胞菌。本研究表明摩拉维亚假单胞菌FP1761具有潜在的植物防病促生功能,但其III型分泌系统与植物益生互作机制有待进一步解析。     

茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析在干旱胁迫下的表达分析

以铁观音茶树叶片为材料,利用逆转录PCR及RACE法,克隆了茶树几丁质酶基因CsChi(GenBank登录号为KR078345).CsChi基因的cDNA全长为1 192 bp,包含972 bp的开放阅读框(ORF),编码323个氨基酸.生物信息学分析结果表明,CsChi蛋白的分子量为34.33 ku;理论等电点pI为8.44;原子组成为C1519H2285N413O464S18,总原子数为4 699;蛋白质结构分析显示该蛋白有6个蛋白的跨膜区域,属于跨膜蛋白;存在于细胞外;没有卷曲螺旋结构存在;CsChi基因编码的蛋白属于糖苷水解酶19家族,含有保守的ChtBD1结构域,与溶菌酶的保守结构域类似,可能兼具几丁质酶活性和溶菌酶活性,qPCR定量分析结果显示在不同干旱胁迫处理下茶树的CsChi基因的表达量,与对照组相比有所增加.推测CsChi基因在茶树干旱等逆境胁迫中起重要作用.     

Changes in the MHC Class II+ Dendritic Cell Population of Ovine Skin In Response to Orf Virus Infection

Abstract— Class II+ dendritic cells were widely distributed throughout normal ovine skin in two main locations: a) in or immediately adjacent to the epidermis and epidermal appendages and b) in the vicinity of the blood vessels. They are unlikely to represent a homogeneous population particularly since Langerhans cells, which previously have been found throughout the epidermal appendages, were located only in the epidermis using acetylcholinesterase staining. Following infection with orf virus, a dense mass of closely associated class II+ dendritic cells develops in the exposed necrotising dermis, adjacent to infected hair follicles and under infected degenerating epidermis. These cells interact and appear to form a barrier to invasion, a framework for immune defence and a template for subsequent epidermal repair; they seem to provide the basis of a highly integrated local dermal defence system. Résumé— Des cellules dendritiques de classe II+étaient largement réparties dans la peau ovine normale dans deux principales zones a) dans l'épiderme et les annexes épidermiques ou dans leur voisnage immédiat b) au voisinage des vaisseaux sanguins. Il est peu probable qu'elles représentant une population homogène particulièrement parce que les cellules de Langerhaps, qui ont été découvertes précédemment dans l'ensemble des annexes épidermiques, furent localisées seulement dans l'épiderme en utilisant une coloration à l'acétylcholinesterase. Après une infection par le virus de l'ecthyma, il se forme une masse dense de cellules dendritiques de classe II+étroitement associées, dans le derme nécrotique atteint, adjacente aux follicules pileux infectés et sous l'épiderme dégénératif infecté. Ces cellules interagissent et apparaissent former une barrière à l'invasion, un cadre pour les défenses immunitaires et un patron pour la réparation épidermique ultérieure; elles semblent fournir les bases d'un système de défense dermique local hautement intégré. Zusammenfassung— Klasse II+-Dendritenzellen waren in der gesamten Haut von normalen Schafen in vorwiegend zwei Bereichen verbreitet: a) in oder unmittelbar neben der Epidermis und der epidermalen Anhangsgebiete und b) in dor Nähe dar Blutgefäße. Sie stellen wahrscheinlich keine homogène Population dar, da die Langerhanszellen, die früher übarall in den epidermalen Anhangsgebilden nachgewiesen wurden, bei Acetylcholinesterase-Färbung nur in der Epidermis zu finden waren. Nach einer Orf-Virus-Infektion formiert sich eine dichte Masse aus eng verbundenen Klasse II+-Dendritenzellen in der betroffenen nekrotisierenden Dermis unmittelbar neben den infizierten Haarfollikeln und unter der infizierten, degenerierenden Epidermis. Diese Zellen stehen untereinander in Verbindung und bilden anscheinend eine Barrière gegen die Invasion, ein Gorüst für die Immunabwehr und einen Ausgangs punkt für die anschließenden Reparaturvorgänge in der Epidermis. Sie scheinen als Basis eines hochentwickelten lokalen Abwehrsystems der Haut zu fungieren. Resumen Células dendríticas de class II+ se observaron en gran cantidad en la piel de la oveja especialmente en dos localizaciones: a) en la epidermis, en la dermis muy próxima a la epidermis y en anejos cutáneos y b) en las proximidades de los vasos sanguíneos. No parece tratarse de una población homogénea de células puesto que las células de Langerhans, que previamente se habían encontrado en los anejos epidérmicos, se encontraron únicamente en la epidermis utilizando técnicas de detección del acetilcol-inesterasa. Después de la infección con el virus del ectima contagioso ovino se observó una masa de células dendríticas de clase II, dispuestas de forma muy densa, en las proximidades de la dermis necrosada y de los folículos pilosos y de la epidermis infectada. Eatas células interaccionan entre si y parecen formar una barrera contra la invasión, una red inmunitaria de defensa y participar en la reparación de la epidermis; parece ser que esta población de células dendriticas son la base de un sistema de defensa dérmico local altamente integrado.     

Evaluation of a radioimmunoassay for type III procollagen peptide in the dog

Type III procollagen peptide (P-3-P) is a serum marker for hepatic fibrosis in humans. The utility of a commercially available radioimmunoassay for P-3-P was evaluated in the dog. The specificity of the assay was assessed by polyacrylamide gel electrophoresis (PAGE) of canine serum and purified bovine P-3-P, followed by Western immunoblotting with rabbit aniti-P-3-P serum. The sensitivity was assessed by performing the radioimmunoassay on dilutions of sera from 22 dogs. Polyacrylamide gel electrophoresis of purified bovine P-3-P and sera from two dogs suspected of having elevated P-3-P concentrations revealed no homologous bands of staining. Western immunoblotting showed marked cross-reactivity of the high antisera concentrations with several components of the serum proteins, but none corresponding to the purified P-3-P. All tested sera from dogs had minimal competitive binding with radiolabeled P-3-P in the radioimmunoassay. Dilution curves of dog sera did not parallel either the standard curve or the dilution curve of a known test human serum. There were no statistically different P-3-P concentrations in any of the groups of dogs studied. It was concluded that currently available radioimmunoassay kits for the measurement of P-3-P in the human are not applicable in the dog. Seemingly, the structure or metabolism of canine P-3-P may vary significantly from that of the bovine or human, limiting the sensitivity and specificity of this assay in the dog.     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

水稻与稻瘟病菌非亲和性互作中重要防御酶活性变化规律研究

 选以CO39为背景的水稻抗稻瘟病近等基因系,与稻瘟菌生理小种ZC₁₃(菌株97-151a)组成的3类典型非亲和性互作,以亲和性互作为对照,对各互作中过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶及β-1,3-葡聚糖酶的活性变化规律进行了系统研究。完全非亲和性互作C101A51/97-151a、高度非亲和性互作C101L AC/97-151a及中度非亲和性互作C104 PKT/97-151a,POD比活性接种后即开始明显升高,48h前达到高峰,升高趋势一直持续到7d完全显症时,幅度基本与各互作非亲和程度呈正相关;亲和性互作CO39/97-151a接种后40 h POD比活性才开始升高,4~6 d达到高峰,峰值也较大。3类非亲和性互作PAL比活性在接种后0 h或16 h开始较明显升高,整个互作中形成3~4个较明显的峰;亲和性互作中PAL比活性一直明显下降。3类非亲和性互作外切几丁质酶比活性接种后即开始升高,基本一直保持升高趋势,在40 h前幅度较大,并形成1~3个较高的峰;亲和性互作外切几丁质酶比活性接种后即开始大幅度升高直至完全显症,48h后幅度远高于非亲和性互作。3类非亲和性互作β-1,3-葡聚糖酶比活性在24 h内开始较明显升高,在48h前形成2~3个较明显的峰;亲和性互作在接种后β-1,3-葡聚糖酶比活性即开始升高,在48h后显著高于非亲和性互作。讨论了POD、PAL、几丁质酶及β-1,3-葡聚糖酶参与水稻抗稻瘟病的可能性。     

转基因水稻对稻瘟病的抗性研究

 采用苗期初筛、复筛、抗谱测定和田间自然诱发试验等不同鉴定方法,对经分子检测证明已整合有碱性几丁质酶基因和β-1,3-葡聚精酶基因的22个转化系的转基因水稻植株进行稻瘟病抗性鉴定研究,筛选出对稻瘟病的抗性比原种对照七丝软占有明显提高的一系列转基因水稻品系,其中表现高抗的有来自F4-9转化株系的7个品系。高抗材料的R7代品系,经室内抗谱测定及田间病圃试验结果,仍然表现高抗稻瘟病。本研究通过转基因技术,成功地将优质感病品种改良成高抗品系,研究结果证明了利用基因工程手段培育抗病水稻新品种是一个非常有希望的育种途径。     

枯草芽孢杆菌G3菌株的抗菌物质及其特性

 产几丁质酶枯草芽孢杆菌G3菌株的固体培养物在黄瓜灰霉病菌和番茄叶霉病菌抑菌试验中证实,抑菌活性物质存在于过滤上清液中,它们是从酸沉淀物中提取出的伊枯草菌素、生物表面活性素和存在于盐析粗蛋白中的几丁质酶。在叶霉孢子萌发试验中,伊枯草菌素微弱地抑制孢子萌发但强烈破坏芽管和新生菌丝;生物表面活性素和几丁质酶则强烈抑制孢子萌发并长久性地抑制芽管伸长。在PDA平板上的灰霉菌丝抑菌试验中,伊枯草菌素抑制菌丝生长,引发菌丝顶端膨大,形成泡囊,泡囊破裂后原生质外泄;几丁质酶抑制菌丝生长,引发产生不规则的菌丝团;生物表面活性素在平皿上对菌丝则不显示出抑菌活性。     

Transgenic cucumber expressing an endogenous class III chitinase gene has reduced symptoms from Botrytis cinerea

A class III chitinase gene (CHI2) is induced in cucumber plants (Cucumis sativa L.) in response to infection by pathogenic microorganisms. Infection of Botrytis cinerea, causal agent of gray mold disease on cucumber, also induces CHI2 expression. To investigate whether CHI2 is involved in resistance to gray mold disease, transgenic cucumber plants were produced to overexpress the CHI2 gene. One line was analyzed in detail in terms of disease resistance. The transgenic cucumber plant (CC2) constitutively expressed CHI2 and reduced the symptoms of B. cinerea for 4 days after inoculation compared with nontransgenic plants. However, this inhibitory effect was not absolute, and CC2 eventually developed serious disease symptoms. Chitinase activity of the crude extract from CC2 leaves was higher than that from nontransgenic plants. A high-molecular-weight fraction containing CHI2 from CC2 leaves had fungistatic activity against B. cinerea. Interestingly, the low-molecular-weight fraction from CC2 leaves with CHI2 removed also had fungistatic activity against B. cinerea. Not only the introduced chitinase activity but also the endogenous defense reactions activated by overexpression of CHI2 may be involved in the enhanced gray mold disease resistance in CC2.