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.     

高能湿法球磨制备鱼胶原肽螯合钙及其理化特征

为了拓展胶原肽螯合钙的制备工艺,以鱼胶原肽和CaCl2为原料,以高能湿法球磨的方法制备了鱼胶原肽螯合钙,并采用FT-IR红外图谱、X-衍射、EDS能谱等技术手段对其理化特性和结构进行分析。结果显示,随着球磨时间从15 min延长到240 min,鱼胶原肽的平均粒径逐渐降低,ZETA电位绝对值、分子量和pH值逐渐增加,鱼胶原肽对钙的螯合率逐渐从74.21%增加到85.42%。鱼胶原肽螯合钙与鱼胶原肽的主体化学结构相似,但是球磨后鱼胶原肽的-COO-1 VAS吸收峰(1 643.89 cm~(-1))向短波数方向移动到1 540～1 555 cm~(-1),而-NH2的特征吸收峰(3 313.9 cm~(-1))向长波数方向移动到3 337.12～3 380.59 cm~(-1)。球磨后鱼胶原在2θ=20°处的特征宽吸收峰消失,而在2θ=26、28、48和58°处出现多个尖峰和弥散峰,表明其从无定形结构向兼具结晶和无定形结构转变。鱼胶原肽螯合钙中钙元素比例逐渐增加,证实鱼胶原肽对钙的螯合率增加。研究表明,高能湿法球磨可应用于鱼胶原肽螯合钙的制备,该方法具有加工工艺简单、无污染和螯合率高等优点。     

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly,Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.     

可可毛色二孢全基因组分泌蛋白的预测及分析

【目的】可可毛色二孢（Lasiodiplodia theobromae）是一种世界性分布的重要植物病原真菌,可引起严重的葡萄溃疡病（Botryosphaeria dieback）,影响果木品质并造成巨大的经济损失。本研究预测并分析可可毛色二孢基因组范围内的分泌蛋白,并明确其基本特征,为该病菌分泌蛋白致病机理的研究打下基础。【方法】依据已公布的可可毛色二孢全基因组序列,利用信号肽预测软件SignalP v5.0、跨膜结构分析软件TMHMM v2.0、细胞器定位分析软件ProtComp v9.0、GPI锚定预测软件big-PI Fungal Predictor和亚细胞器定位分析软件TargetP v2.0生物信息学软件对该菌中的典型分泌蛋白进行筛选。对分泌蛋白N端信号肽的长度、氨基酸使用频率及其切割位点进行统计分析。依据蛋白序列的同源性,应用BLASTP程序对分泌组蛋白进行功能注释分析,预测其生物学功能。采用蔗糖酶缺陷的酵母分泌系统,对所选分泌蛋白的信号肽进行活性检测。利用qRT-PCR方法检测所选分泌蛋白基因在可可毛色二孢侵染葡萄中的表达情况。【结果】在可可毛色二孢全基因组编码蛋白中共筛选获得552个潜在的具有典型信号肽的分泌蛋白,占全基因组预测蛋白总数的4.3%,其编码蛋白长度集中于101—400 aa。信号肽统计分析表明,其信号肽长度以18—20 aa的序列最为集中,信号肽长度为20 aa的蛋白数量最多。信号肽中使用频率最高的氨基酸为丙氨酸;非极性、疏水的氨基酸使用频率最高,占氨基酸总数的60.2%。其信号肽的-3至-1位置上的氨基酸相对保守,切割位点属于A-X-A类型,可被Sp I型信号肽酶识别并切割。336个分泌蛋白具有功能注释,其功能较多集中于细胞壁降解有关的酶类以及致病相关蛋白,并且这些蛋白在分子量、等电点、脂肪族氨基酸指数等方面均存在差异。通过蔗糖酶缺陷的酵母分泌系统证实,挑选的9个分泌蛋白信号肽均具有分泌活性。qRT-PCR检测结果表明,所选分泌蛋白基因在该病菌侵染初期的表达发生变化。【结论】利用生物信息学分析技术从可可毛色二孢全基因组中共预测获得552个经典分泌蛋白。其信号肽氨基酸长度分布广泛,氨基酸组成中非极性、疏水的氨基酸使用频率最高。功能注释主要集中在细胞壁组分降解相关的酶类、致病侵染相关的坏死诱导相关蛋白以及几丁质结合蛋白等。     

Modulation of plant root traits by nitrogen and phosphate: transporters,long-distance signaling proteins and peptides,and potential artificial traps

As sessile organisms, plants rely on their roots for anchorage and uptake of water and nutrients. Plant root is an organ showing extensive morphological and metabolic plasticity in response to diverse environmental stimuli including nitrogen (N) and phosphorus (P) nutrition/stresses. N and P are two essential macronutrients serving as not only cell structural components but also local and systemic signals triggering root acclimatory responses. Here, we mainly focused on the current advances on root responses to N and P nutrition/stresses regarding transporters as well as long-distance mobile proteins and peptides, which largely represent local and systemic regulators, respectively. Moreover, we exemplified some of the potential pitfalls in experimental design, which has been routinely adopted for decades. These commonly accepted methods may help researchers gain fundamental mechanistic insights into plant intrinsic responses, yet the output might lack strong relevance to the real situation in the context of natural and agricultural ecosystems. On this basis, we further discuss the established—and yet to be validated—improvements in experimental design, aiming at interpreting the data obtained under laboratory conditions in a more practical view.     

大熊猫源枝孢样枝孢霉野生株(Z20)与突变株(Zt)部分生物学特性及药敏试验比较

为探明枝孢样枝孢霉野生株Z20与突变株Zt在生物学特性及药物敏感上的差异,将枝孢样枝孢霉Z20和Zt于PDA培养基上连续培养7 d,每天观察菌落形态及长势。将菌株Z20和Zt在沙氏液基上培养4 d后用透射电镜对其超微结构进行观察。采用硫酸-苯酚法测定枝孢样枝孢霉Z20和Zt的菌丝体多糖含量。运用96孔板微量稀释法测定5种常见抗真菌药物对枝孢样枝孢霉Z20和Zt的最小抑菌浓度。结果表明:在PDA培养基上菌株Zt的生长速率高于Z20,菌落颜色较浅,孢子数量减少。在透射电镜下,野生株Z20的菌丝壁厚度和菌丝宽度显著大于突变株Zt,且菌丝的含糖量也显著高于突变株Zt。药敏试验中,除灰黄霉素外,枝孢样枝孢霉Zt对5种药物的敏感性均高于Z20。     

寡聚酸碘抑制TMV活性测定及对番茄生长影响初探

为研究新型壳寡糖衍生物类农用制剂寡聚酸碘(寡聚N-乙酰基-D-氨基葡萄糖醛酸碘)对烟草花叶病毒(TMV)的抑制作用及对番茄生长、生理特性的影响,以番茄品种:L402、贝鲁特、粉多利作为供试材料,以氨基寡糖素及50%腐霉利L可湿粉剂作为对比,通过室内生测试验试验,测定寡聚酸碘对番茄病毒病中常见病原菌TMV的抑制效果、对番茄生长及及番茄幼苗茎尖中吲哚乙酸、赤霉素及叶绿素含量的影响。寡聚酸碘对TMV的抑制效果表现出良好的速效性和持效性,接种后17d,抑制效果从45.08%迅速提升到74.48%,接种21~35d,对TMV的抑制效果维持在80%以上。寡聚酸碘对TMV的EC50值为15.62mg/L,对TMV的抑制活性高于3%氨基寡糖素水剂寡聚酸碘处理,有效成分300.0mg/L的寡聚酸碘可作为抑制TMV活性的最优供试剂量;寡聚酸碘在300~1 200mg/L处理剂量下,对供试的3个番茄品种(L402、贝鲁特、粉多利)安全,未见任何药害症状,与CK、3%氨基寡糖素及50%腐霉利L可湿粉剂相比,可以明显提升番茄幼苗株高、茎尖内IAA、GA含量及叶片中总叶绿素含量。寡聚酸碘处理的番茄茎秆粗壮,叶色浓绿,生长性状良好。因此,寡聚酸碘具有抑制TMV活性及促进番茄生长的双重功效,开发利用前景良好。     

Chemical composition of Lippia spp. essential oil and antimicrobial activity against Aeromonas hydrophila

Phytotherapy can replace antibiotic administration as an alternative to control Aeromonas hydrophila, one of the main bacteria involved in the aetiology of farmed fish diseases. Given that plants of the Lippia spp. genus show biological potential for antimicrobial activity, this study evaluated the chemical composition of essential oils extracted from Lippia alba, Lippia origanoides and Lippia sidoides and their activity against A. hydrophila. The oils were obtained by steam distillation in a Clevenger‐type apparatus and their composition determined by gas chromatography and mass spectrometry (CG/MS). Antibacterial activity was assessed by calculating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using broth microdilution method. The main compounds identified were geranial (25.4%) and neral (16.6%) in L. alba oil, carvacrol (40.4%) and p‐cymene (11.4%) in L. origanoides oil and thymol (76.6%) and ortho‐cymene (6.3%) in L. sidoides oil. The three Lippia species showed bacteriostatic and bactericidal action against A. hydrophila, with MICs and MBCs ranging from 1250 to 5000 μg mL^?1. Of the species tested, the best performance was obtained with essential oil of L. sidoides.     

Characterization of Collagen Enzymatic Hydrolysates Derived from Lizardfish (Synodus fuscus) Scales

Acid soluble collagen was extracted from the scales of lizardfish and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis. After 8 h of collagen hydrolysis, hydrolysates had an estimated degree of hydrolysis (DH) from 4 ± 0.05 to 25 ± 0.63% (p < 0.05). All hydrolysates had angiotensin converting enzyme inhibitory and antioxidant activity. These activity levels showed little change after treatment with gastrointestinal proteases. Results indicated that the lizardfish by-products may be improved by enzymatic treatment with acid-soluble collagen from lizardfish scales.