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.     

The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

A kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.     

中药成分辛弗林对胶体金免疫层析法检测β-受体激动剂的影响

旨在探究陈皮、青皮、木瓜、厚朴、红景天等5种中药材提取液以及饲喂了陈皮和青皮的猪尿液胶体金免疫层析法检测结果呈阳性的原因。建立测定猪尿及中药提取物中辛弗林的LC-MS/MS检测方法，测定陈皮、青皮的提取物和饲喂陈皮、青皮后猪尿中的辛弗林浓度。用小鼠肝微粒体孵育CGIA检测呈阳性的陈皮、青皮、木瓜和厚朴4味中药提取物。饲喂陈皮和青皮后的猪尿液检出辛弗林，浓度分别为1.36和1.65 μg·mL^-1；在陈皮和青皮的提取复溶液中检出辛弗林，浓度分别为132.6和312.7 μg·mL^-1。厚朴和木瓜两种中药提取物经过肝微粒体孵育后，在2~5 h逐渐由CGIA检测阳性转为阴性，陈皮和青皮孵育后，0~6 h的结果均为CGIA检测阳性；阴性中药组CGIA检测均呈阴性，而添加辛弗林的阳性对照组0~6 h CGIA检测均呈阳性。猪较大剂量使用陈皮和青皮会引起猪尿β-受体激动剂CGIA检测出现假阳性，该假阳性现象跟中药成分辛弗林有关，体外试验中辛弗林不易被小鼠肝微粒体代谢。     

油松谷胱苷肽转移酶Tau1单体结构稳定性研究

[目的]探究油松PtGSTU1结构与功能的关系,探讨油松PtGSTU1蛋白的单体稳定性。[方法]利用同源建模模拟PtGSTU1的三维结构,推测其N末端18位精氨酸(Arg18)和C末端103位天冬氨酸(Asp103)能够形成氢键来稳定蛋白单体结构。利用定点突变,分别将Arg18和Asp103突变为具有不同极性和构象的氨基酸残基,检测其蛋白的催化活性及结构稳定性。[结果]6个Arg18突变体均无法获得高纯度的具有正确折叠的可溶蛋白,而Asp103突变体可以表达为可溶蛋白,Asp103突变体对不同底物的催化活性和亲和力明显低于野生型,对经典底物CDNB和GSH反应的催化速率(Vmax)降低了至少9倍,对底物的催化效率(kcat/Km)也明显降低。[结论]Arg18和Asp103之间形成的氢键对稳定PtGSTU1单体结构具有重要作用,由于植物GST蛋白N端的保守性和C端结构域的多变性,Arg18的突变对结构和活性的影响大于Asp103,同时预示着C端结构域中可能存在其他氨基酸位点能够与18位精氨酸形成氢键,从而稳定蛋白单体折叠结构。     

华北地区十字花科芸薹属蔬菜上立枯丝核菌的病原生物学研究

 由丝核菌引起的十字花科蔬菜叶腐和茎基腐病在中国华北地区普遍发生,其中以河北、内蒙以及北京较为严重。2011~2018年,从华北地区不同省份具有典型叶腐和茎基腐症状的芸苔属蔬菜上分离获得95个丝核菌(Rhizoctonia spp.)分离物,大多数分离自发病植株的叶部,少数分离自茎基部。通过细胞核染色,87株菌属于多核丝核菌,另外8株属于双核丝核菌;经菌丝融合鉴定、rDNA-ITS区及TEF-1α(translation elongation factor 1-alpha, TEF-1α)序列分析,大多数多核丝核菌属于立枯丝核菌(Rhizoctonia solani)AG-2-1(74％),其他少数分别属于AG-1-IB(16％)、AG-4-HG II(2％)和双核丝核菌AG-A(8％)。温室条件下进行寄主范围致病力测定,各分离物对原寄主都表现出致病力,呈现典型叶腐或茎基腐症状;对其他作物的致病力差异较大。不同融合群(Anastomosis group,AG)的菌株对寄主致病力大小存在差异,AG-2-1致病力最强,只有AG-A对叶部没有致病力。AG-2-1对寄主叶部的致病力和对茎基部的致病力呈显著正相关,AG-1-IB对寄主叶部的致病力和对茎基部的致病力无显著相关性。     

A pilot study to investigate the measurement of immunoglobulin A in Welsh Cob and Welsh Pony foals’ faeces and their dam’s milk

ABSTRACT

Aims: To determine if an ELISA for measurement of IgA in equine serum could be used to measure concentrations of IgA in foal faeces and to determine correlations with concentrations in the milk of the dam.

Methods: Faeces from 20 Welsh Cob and Welsh Pony foals and milk from their dams were collected within 12?hours (Day 0) and at 6 days after parturition (Day 6). On Day 6, faeces could not be collected from 2/20 foals, and milk samples could not be collected from 3/20 mares. An equine IgA ELISA validated for serum and plasma was used to measure concentrations of IgA in all samples in triplicate. The precision of the assay for each sample type was determined using modified CV.

Results: IgA was not detectable in 7/20 Day 0 faecal samples and in 2/18 Day 6 faecal samples. For samples with detectable IgA, the mean modified CV was 10.5 (95% CI?=?6.0–15.0)% for Day 0 faecal samples, and was 6.8 (95% CI?=?4.3–9.4)% for Day 6 faecal samples. Median concentrations of IgA in faeces on Day 0 were lower than concentrations on Day 6 (0.7?mg/g vs. 37?mg/g dry matter; p?=?0.003). Concentrations of IgA in milk and faeces on Day 6 were statistically correlated (r?=?0.59; p?=?0.006).

Conclusions and clinical relevance: The IgA ELISA showed acceptable precision when used to estimate concentrations of IgA in foal faeces during the first week of life, but IgA could not be detected in 37% of meconium samples collected on Day 0. This assay may be useful for investigation of the role of maternal milk IgA in the gastrointestinal tract of neonatal foals, but further assessment of both accuracy and precision of the ELISA is required.     

miR-106b-5p靶向KLF4调控山羊肌内前体脂肪细胞分化

旨在明确miR-106b-5p对山羊肌内前体脂肪细胞分化的影响，并确定这种作用是通过靶向KLF4来实现的。本研究利用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）技术检测miR-106b-5p在山羊肌内前体脂肪细胞分化过程中的表达模式，通过脂质体转染技术将miR-106b-5p mimic和miR-106b-5p inhibitor转入体外培养的山羊肌内前体脂肪细胞，油红O染色法从形态学验证miR-106b-5p对脂肪细胞中脂滴积聚的影响，qRT-PCR检测预测的靶标基因KLF4和脂肪分化标志基因的表达情况，利用双荧光素酶报告系统鉴定miR-106b-5p与KLF4的靶标关系。qRT-PCR结果显示，miR-106b-5p在山羊肌内前体脂肪细胞诱导分化第3天时表达量最高。在山羊肌内脂肪细胞中干扰miR-106b-5p后油红O染色显示脂滴聚积减少，过表达miR-106b-5p后脂滴聚积增加。在山羊肌内前体脂肪细胞中转染miR-106b-5p inhibitor后PPARγ表达量显著降低（P<0.05），而KLF4表达量极显著升高（P<0.01）；转染miR-106b-5p mimic后LPL和PPARγ表达量极显著升高（P<0.01）。荧光素酶活性试验结果显示，过表达miR-106b-5p可显著抑制KLF4荧光活性。miR-106b-5p通过靶向并负调节KLF4的表达促进山羊肌内脂肪细胞分化。     

Rapid detection of potato late blight using a loop-mediated isothermal amplification assay

Potato late blight caused by Phytophthora infestans is one of the most destructive plant diseases that threaten global food security. Early and effective diagnosis of P. infestans is required before disease management decisions are made. Here, we developed a quick protocol to detect P. infestans based on a loop-mediated isothermal amplification(LAMP) assay. The P. infestans specific multiple copy DNA sequences(PiSMC), a transposon-like element, provides an ideal target for molecular detection of this pathogen. We designed highly specific and sensitive primers allowing effective LAMP detection of the pathogen at 64℃ in 70 min. In the validation assay, all 15 P. infestans isolates collected from China, Europe and South America could be positively detected, but results of the other 9 Phytophthora species infecting different plants, fungal and bacterial plant pathogens tested were negative. The detection limit of this assay is 1 pg P. infestans DNA. Moreover, the LAMP-PiSMC assay is able to detect P. infestans from infected leaves, tubers and soil. Taken together, this study reports the development of a specific and sensitive LAMP-PiSMC assay for early diagnosis of potato late blight.     

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.     

用于水生病原菌的3种高通量活菌计数方法的比较

以一株副溶血弧菌(Vibrio parahaemolyticus)作为研究对象，比较了MTT [3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]比色法、ATP生物发光法和高通量生长曲线法在活细菌高通量计数上的应用效果。用96孔培养板进行不同浓度细菌活菌计数，确定了上述3种方法在副溶血弧菌活菌计数的标准曲线和线性范围。结果显示，副溶血弧菌的MTT比色法以DMSO溶解的MTT产物甲瓒在555 nm的吸光度(OD555 nm)为计数依据，活菌数的对数(LgC)与LgOD555 nm线性关系的标准曲线为LgC=(1.0439±0.0200)LgOD555 nm+(8.0565±0.0125)，相关系数R²=0.9965，线性检测范围为7.8×106~2.5×108 CFU/ml；ATP生物发光法以ATP产生的相对发光度值(RLU)为计数依据，LgC与LgRUL线性关系的标准曲线为LgC=(0.9590±0.0065)LgRLU+(0.9949±0.0366)，相关系数R²=0.9994，线性检测范围为1.0×104~3.0×108 CFU/ml；高通量生长曲线法以生长曲线达到拐点的时间(Ts)为计数依据，LgC与Ts线性关系的标准曲线为LgC=?(0.8727±0.0230)Ts+(9.0128±0.1572)，相关系数R²=0.9924，线性检测范围为1.0×100~1.0×107 CFU/ml。用3种方法对实际菌液测量并与平板计数法比较表明，ATP生物发光法与高通量生长曲线法有很好的准确性，MTT比色法准确度稍差，而高通量生长曲线法有最宽的线性范围，也最适合高通量测定。