转中国对虾抗菌肽基因水稻抗白叶枯病效应初析

抗菌肽是一类具有抗菌活性的多肽类物质。利用农杆菌介导法将中国对虾抗菌肽基因np3和np5分别导入粳稻品种爱知旭中,PCR检测显示,T0代转np3基因和np5基因水稻植株的阳性率分别为36%和45%。RT-PCR检测T1代转基因水稻,结果表明抗菌肽基因在RNA水平上得到表达,而抗菌肽的表达对农艺性状未产生显著影响。用白叶枯病菌小种CR4分别对4个T1代转np3基因和np5基因株系进行接种,所有转基因株系均表现出比对照更高水平的抗性。为了验证抗菌肽基因的广谱抗性,进一步检测了np5转基因株系对3个菌株JS97-2、ZHE173和OS-225的抗性,发现中国对虾抗菌肽基因np5对白叶枯病菌可能具有较广谱的抗性。     

Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

Characterization of Streptomyces spp. Isolated from the Sea Surface Microlayer in the Trondheim Fjord, Norway

The water surface microlayer is still poorly explored, although it has been shown to contain a high density of metabolically active bacteria, often called bacterioneuston. Actinomycetes from the surface microlayer in the Trondheim fjord, Norway, have been isolated and characterized. A total of 217 isolates from two separate samples morphologically resembling the genus Streptomyces have been further investigated in this study. Antimicrobial assays showed that about 80% of the isolates exhibited antagonistic activity against non-filamentous fungus, Gram-negative, and Gram-positive bacteria. Based on the macroscopic analyses and inhibition patterns from the antimicrobial assays, the sub-grouping of isolates was performed. Partial 16S rDNAs from the candidates from each subgroup were sequenced and phylogenetic analysis performed. 7 isolates with identical 16S rDNA sequences were further studied for the presence of PKS type I genes. Sequencing and phylogenetic analysis of the PKS gene fragments revealed that horizontal gene transfer between closely related species might have taken place. Identification of unique PKS genes in these isolates implies that de-replication can not be performed based solely on the 16S rDNA sequences. The results obtained in this study suggest that streptomycetes from the neuston population may be an interesting source for discovery of new antimicrobial agents.     

Effect of changing coating process parameters in the preparation of antimicrobial-coated silk sutures: An in vitro study

Braided silk sutures were coated using a combination of poly(ε-caprolactone) (PCL) and sulfamethoxazole trimethroprim (SMZ) to investigate their antimicrobial performance. SMZ (2500 μg/ml) was combined with different PCL concentrations, i.e., 2.5 %, 5 %, 7.5 %, and 10 % (w/v). Antimicrobial test results showed that SMZ and PCL-treated silk sutures exhibited increasing antimicrobial efficacy against Gram-negative (Escherichia coli) and -positive bacteria (Staphylococcus aureus) with increasing PCL concentrations. The tensile and knot strength of sutures coated with 10 % PCL were significantly higher than those of sutures coated with 7.5 %, 5 %, and 2.5 % PCL. Treatment with PCL exhibited a positive effect on drug release from the sutures. Significant traits of antibacterial activity were observed up to 4 days after instalment of 10 % PCL-coated silk sutures. Under a scanning electron microscope, untreated silk sutures showed a surface heavily coated with bacteria, whereas treated sutures showed a smooth surface without bacteria. The results of this study indicate that SMZ combined with high concentrations of PCL may afford a suitable antibacterial coating agent for braided silk sutures.     

水稻不育系系谱抗稻瘟病基因同源序列的克隆与序列分析

根据已知NBS-LRR类抗病基因结构中氨基酸的保守区域设计简并引物,对水稻不育系系谱NBS-LRR类抗病基因同源序列进行克隆、测序和聚类。同源序列克隆与分析表明,7个水稻不育系系谱亲本中共获得14个阳性克隆,其中11个含有NBS-LRR类抗病基因所特有的保守氨基酸结构域KinaseⅠ、KinaseⅡ、KinaseⅢ及跨膜区域。这些片段间同源性最高达98.3%,最低仅为29.7%;11个氨基酸片段与抗病基因Xal（AB002266）的同源性较高,42%-45%的氨基酸序列相同,59%-64%的氨基酸序列相似,且与水稻其他NBS-LRR类抗性蛋白相似性很高。通过聚类分析,可以将其分为3类,分别记为G1、G2、G3,并发现抗源谷农13所含有的G3类基因,通过天谷、福伊传给后代谷丰、全丰。     

甘蓝型油菜DH群体种子活力性状的遗传分析

高种子活力是决定后期植株优良形态建立和作物高产的基础。为明确甘蓝型油菜种子活力的遗传规律,本研究选用两个发芽势差异较大的甘蓝型油菜中双11号和R11配制杂交组合,构建了包含280个株系的甘蓝型油菜DH群体。利用建立的室内种子萌发检测系统,对4个种子活力评价指标,包含萌发曲线下面积（area underthe germination curve AUC)、平均发芽时间（mean germination time MGT)、发芽率达到50%所用的时间（time taken for 50% seeds to germinate, T50）、发芽率从16%至84%所用的时间（time between 16% and 84% germination, U8416）,采用主基因+多基因混合遗传模型及偏度和峰度分析方法对种子活力评价指标进行遗传分析。结果表明,萌发曲线下面积受3对主基因+9对微效多基因控制,决定该性状的基因间存在着重叠作用,主基因的遗传率达74.88%,受环境影响较小;平均发芽时间和T50均受2对主基因+微效多基因控制,微效基因的数目分别为13和14对,主基因的遗传率分别为64.20%和64.53%,受环境影响偏大;U8416受4对主基因+8对微效多基因的控制,4对主基因的加性效应均表现为正效应,且决定该性状的基因间存在着互补作用,表明当这些基因同时存在时,基因的加性效应可以增强。主基因遗传率高达87.57%,表明主基因的遗传效应明显,受环境影响较小。研究表明,决定种子活力水平各萌发性状均是由主基因+多基因调控,并且各萌发性状的基因间均表现互作效应,为了使互作的基因间有更多的重组机会,在低世代的选择强度均不宜太大。     

Study of structure-activity relationship in Aurein 1.2 analogs

Two new analogs of Aurein 1.2 antimicrobial peptide were synthesized and the antimicrobial activities were investigated. The results showed that the activity of G1R/F3W analog was higher than the native peptide and the F3W analog. Circular dichroism studies also showed that the secondary structure of the F3W was concentration-dependent, whereas, there was no such relationship seen in the case of G1R/F3W analog. It has been proposed that G1R/F3W activity was based on a single mechanism (snorkeling), while Aurein 1.2 and F3W utilized the snorkeling mechanism at low concentrations (0-0.01 mM) and the carpet mechanism at higher concentrations (0.01-0.1 mM). This study suggests that one pay attention to the concentration of biomolecules in peptide-based drug design.     

ACE Inhibitory Peptide from Skin Collagen Hydrolysate of Takifugu bimaculatus as Potential for Protecting HUVECs Injury

Angiotensin-I-converting enzyme (ACE) is a crucial enzyme or receptor that catalyzes the generation of potent vasopressor angiotensin II (Ang II). ACE inhibitory peptides from fish showed effective ACE inhibitory activity. In this study, we reported an ACE inhibitory peptide from Takifugu bimaculatus (T. bimaculatus), which was obtained by molecular docking with acid-soluble collagen (ASC) hydrolysate of T. bimaculatus. The antihypertensive effects and potential mechanism were conducted using Ang-II-induced human umbilical vein endothelial cells (HUVECs) as a model. The results showed that FNLRMQ alleviated the viability and facilitated apoptosis of Ang-II-induced HUVECs. Further research suggested that FNLRMQ may protect Ang-II-induced endothelial injury by regulating Nrf2/HO-1 and PI3K/Akt/eNOS signaling pathways. This study, herein, reveals that collagen peptide FNLRMQ could be used as a potential candidate compound for antihypertensive treatment, and could provide scientific evidence for the high-value utilization of marine resources including T. bimaculatus.     

Evaluation of comfort properties of polyester knitted spacer fabrics finished with water repellent and antimicrobial agents

In order to impart barrier properties against water and microorganisms on breathable three dimensional spacer fabrics as medical or technical textiles, fabric samples were treated with two water repellent agents and a quaternary ammonium salt namely cetyltrimethylammonium bromide (CTAB), using pad-dry-cure method. Two different water repellent agents based on hydrocarbon and acrylic copolymer were used. The water repellent property of samples was tested by Bundesmann and contact angle tests. Antimicrobial activity of samples was analyzed quantitatively according to AATCC 100. Simultaneous finishing of samples was done with 3 % CTAB and 4 % fluoroalkyl acrylic copolymer. To study the effect of various treatments on comfort related properties, air and water vapor permeability, water repellency and compression were measured. The results showed that the antimicrobial and water repellent spacer fabrics can be achieved applying selected material without significant changes on their comfort properties. Also a regression model was presented to predict the water vapour permeability of knitted spacer fabrics based on course density (CPC) changing.     

大豆抗孢囊线虫病新品种选育及遗传机制研究

以辽豆１０号（感病）为母本,Ｆｒａｎｋｌｉｎｇ（抗病）为父本进行有性杂交,经过５年挖根鉴定和移栽选选择,选育出在疫区比对照品种增产２５％以上、高抗１号、３号上种品系各５个。遗传分析表明,在辽豆１０遗传背景下,北京小黑豆对３号生理小种的抗性是由１个显笥基因和２个隐性基因控制的。生理小种鉴定结果,辽宁有１号和３号两个生理小种,吉林有１号、３号和５号三个生理小种。     

Large-Scale Plasma Peptidomic Profiling Reveals a Novel,Nontoxic, Crassostrea hongkongensis-Derived Antimicrobial Peptide against Foodborne Pathogens

Antimicrobial peptides are a fundamental component of mollusks’ defense systems, though they remain a thinly investigated subject. Here, infection by Vibrio parahemolyticus triggered a significant increase in antimicrobial activity in oyster plasma. By using PBS-challenged oysters as a control, plasma peptides from immunologically challenged oysters were subjected to peptidomic profiling and in silico data mining to identify bioactive peptides. Thirty-five identified plasma peptides were up-regulated post infection, among which, six up-regulated peptides (URPs) showed a relatively high positive charge. URP20 was validated with significant antibacterial activity. Virtually, URP20 triggered aggregation of bacterial cells, accompanied by their membrane permeabilization. Interestingly, URP20 was found to be active against Gram-positive and Gram-negative foodborne pathogens as well as Candida albicans, with no cytotoxicity to mammalian cells and mice. Our study provides the first large-scale plasma peptidomic dataset that identifies novel bioactive peptides in marine mollusks. Further exploration of peptide diversity in marine invertebrates should prove a fruitful pursuit for designing novel AMPs with broad applications.     

甘蓝型油菜DH群体抗裂角性的遗传分析

提高抗裂角性是培育适合机械化收获油菜品种的基本要求。为了明确油菜抗裂角性的遗传规律,本研究利用抗裂角性差异显著的两个甘蓝型油菜品系配制杂交组合,通过小孢子培养构建了一个双单倍体(doubled haploid,DH)群体。采用随机碰撞法对该群体进行连续两年的抗裂角性鉴定,并利用植物数量性状的主基因+多基因混合遗传模型及偏度和峰度分析对抗裂角性进行遗传分析。结果表明,甘蓝型油菜的抗裂角性受3对主基因+多基因控制,涉及的基因数目为8~10对,存在基因的加性效应及基因间的互补作用;两年试验中抗裂角性的主基因遗传率均大于85.00%,主基因遗传率较高,表明该性状主要受主基因控制,受其它微效多基因及环境的影响有限。因此,在油菜抗裂角性遗传改良中,应通过杂交聚合不同抗性等级材料中的主效抗裂角基因,在早期世代加强对抗性的选择,并可以忽略环境因素的影响。     

农杆菌介导的转高赖氨酸蛋白基因（sb401）水稻T4代分析

通过农杆菌介导的方法用富赖氨酸蛋白基因（sb401）转化粳稻品种日本晴,获得了独立的10个转化株系,对转化株系进行连续自交,通过筛选得到9个纯合的T4代转化株系。 Southern blotting分析发现,整合位点是随机的,并为低拷贝（1~3个）。TAI-PCR扩增得到8个T-DNA侧翼序列,并定位于日本晴的7条染色体上。蛋白质和氨基酸测定分析发现,sb401基因对各株系的蛋白质、赖氨酸和其他氨基酸组分的提高起到了一定的作用。将杂交结果与T-DNA插入位置结合分析发现,在低拷贝的情况下,表达量的差异不明显。     

Expression of the Antimicrobial Peptide Piscidin 1 and Neuropeptides in Fish Gill and Skin: A Potential Participation in Neuro-Immune Interaction

Antimicrobial peptides (AMPs) are found widespread in nature and possess antimicrobial and immunomodulatory activities. Due to their multifunctional properties, these peptides are a focus of growing body of interest and have been characterized in several fish species. Due to their similarities in amino-acid composition and amphipathic design, it has been suggested that neuropeptides may be directly involved in the innate immune response against pathogen intruders. In this review, we report the molecular characterization of the fish-specific AMP piscidin1, the production of an antibody raised against this peptide and the immunohistochemical identification of this peptide and enkephalins in the neuroepithelial cells (NECs) in the gill of several teleost fish species living in different habitats. In spite of the abundant literature on Piscidin1, the biological role of this peptide in fish visceral organs remains poorly explored, as well as the role of the neuropeptides in neuroimmune interaction in fish. The NECs, by their role as sensors of hypoxia changes in the external environments, in combination with their endocrine nature and secretion of immunomodulatory substances would influence various types of immune cells that contain piscidin, such as mast cells and eosinophils, both showing interaction with the nervous system. The discovery of piscidins in the gill and skin, their diversity and their role in the regulation of immune response will lead to better selection of these immunomodulatory molecules as drug targets to retain antimicrobial barrier function and for aquaculture therapy in the future.     

柚芽变株系类胡萝卜素代谢差异基因的转录组学分析

为了研究‘琯溪蜜柚’果实红肉突变株系类胡萝卜素代谢和芽变的分子机制,本研究以‘琯溪蜜柚’及其芽变一代品种‘红肉蜜柚’和芽变二代品种‘三红蜜柚’果实为材料,使用RNA-seq技术研究3个品种3个发育时期果肉类胡萝卜素的代谢。经生物信息学分析,共筛选出参与类胡萝卜素生物合成显著差异表达的10个基因,分别为八氢番茄红素合成酶基因（PSY）、六氢番茄红素脱氢酶基因（ZDS）、类胡萝卜素异构酶基因（CRTISO1、CRTISO2）、紫黄质脱环氧酶基因（NPQ1）、9-顺式-环氧类胡萝卜素双加氧酶基因（NCED3、NCED5）、脱落醛氧化酶基因（AAO3）、ABA 8’-羟化酶基因（CYP707A1）、类胡萝卜素裂解双加氧酶基因（CCD4）。共表达分析揭示了7个家族转录因子的表达模式与类胡萝卜素合成基因高度相关,分别为MADS、bZIP、bHLH、MYB、AP2/ERF、NAC和WRKY。通过实时荧光定量PCR（qRT-PCR）验证了部分基因的表达模式,结果与RNA-seq结果高度一致。此外,经对‘红肉蜜柚’果肉转色期的初生和次生代谢通路DEGs富集分析,发现类胡萝卜素的积累过程中,细胞壁、脂质、类黄酮代谢差异基因大量富集。研究结果揭示,‘琯溪蜜柚’果实红肉突变品种‘红肉蜜柚’和‘三红蜜柚’果实类胡萝卜素的形成和积累,是由类胡萝卜素的代谢相关酶基因差异表达和转录因子调控引起的;在果肉红色芽变的同时,伴随着与汁胞粒化相关的基因突变。这对进一步开展柚子果实类胡萝卜素的形成与积累、汁胞粒化研究,为解析柚果实不同颜色突变、汁胞粒化的机制有重要参考价值。     

柑桔栽培品种高效基因转化新技术研究

采用试管实生苗去芽创伤－农杆菌感染法,将人工合成的抗菌肽基因转入柑桔栽培品种,利用后期筛选和Ｐｅｒａｆｉｌｍ包缠法,提高了转化效率。拟转化植株的叶圆片抗Ｋａｎ能力分析结果表明,锦橙、新会橙和沙田袖的转化效率分别达到４６．３％,３９．５％和４３．７％。根据对 Ｋａｎ阳性植株的ＰＣＲ分析,证明有抗菌肽基因出现。结果表明,这是一种简便、高效、重复性好的实用基因转化方法。     

Isolation of Sesquiterpenoids and Steroids from the Soft Coral Sinularia brassica and Determination of Their Absolute Configuration

Two undescribed rearranged cadinane-type sesquiterpenoids (1–2), named sinulaketol A-B, together with one new chlorinated steroid (3), one new gorgosterol (4), one known sesquiterpene (5), one known dibromoditerpene (6) and two known polyhydroxylated steroids (7–8) were isolated from the soft coral Sinularia brassica. The structures of these compounds were established by extensive spectroscopic analysis, including HRESIMS, 1D, and 2D NMR spectroscopy. Their absolute configurations were also determined by the ECD calculations and DP4+ probability analysis. Antileishmanial activity of compounds 1–8 was evaluated in vitro against the amastigote forms of Leishmania donovani, in which compounds 3, 6, and 7 inhibited the growth of L. donovani by 58.7, 74.3, 54.7%, respectively, at a concentration of 50 μM. Antimicrobial effect of the isolated compounds were also evaluated against Candida albicans, Staphylococcus aureus, and Escherichia coli. Compound 6, a brominated diterpene, exhibited antimicrobial effect against S. aureus.     

油菜miR156基因家族及其靶基因生物信息学分析及鉴定

为阐明油菜miR156基因家族的进化过程和靶基因作用位点,分析了油菜miR156基因家族的序列特征、进化模式和候选靶基因。共获得35个bna-miR156基因,对应44个成熟序列。这些bna-miR156基因分布在油菜的17条染色体上,其中C3染色体上分布着5个bna-miR156基因。bna-miR156基因在产生成熟序列的区域高度保守,而其它其它位置保守性较低,进化分析结果显示bna-miR156家族可以分为5个分支,家族成员分布较为分散,且位于同一条染色体上的bna-miR156基因并没有表现出更近的亲缘关系。另外,系统发育分析结果显示SBP-box基因在双子叶植物油菜、拟南芥中存在相似的进化模式,进一步发现只有部分BnaSBP家族基因拥有bna-miR156的作用位点,其中30个BnaSBP基因的靶位点位于编码区,11个位于3`UTR。一些拥有bna-miR156作用位点的BnaSBP具有组织表达特异性,因此bna-miR156可能与BnaSBP相互作用,调控油菜生长发育过程。      

Review of immobilized antimicrobial agents and methods for testing

Antimicrobial surfaces for food and medical applications have historically involved antimicrobial coatings that elute biocides for effective kill in solution or at surfaces. However, recent efforts have focused on immobilized antimicrobial agents in order to avoid toxicity and the compatibility and reservoir limitations common to elutable agents. This review critically examines the assorted antimicrobial agents reported to have been immobilized, with an emphasis on the interpretation of antimicrobial testing as it pertains to discriminating between eluting and immobilized agents. Immobilization techniques and modes of antimicrobial action are also discussed.