基于Cfku70基因失活策略构建果生刺盘孢的高效基因敲除底盘菌株

果生刺盘孢侵染危害多种植物,是重要的植物病原真菌。在一些丝状真菌中,敲除非同源末端连接修复通路的关键基因ku70或ku80可显著提高同源重组效率,进而提高靶基因置换频率。本研究从果生刺盘孢基因组鉴定到Cfku70和Cfku80两个基因,并明确了基因失活对菌株生物学表型和基因敲除效率的影响。敲除Cfku70或Cfku80不影响菌株的菌落形态、营养生长、产孢、分生孢子萌发、侵染结构发育和致病;Cfku70基因敲除还大幅提升3个测试基因的敲除效率。本研究证实Cfku70基因失活能显著提高果生刺盘孢的基因敲除效率,适宜作为高效基因敲除的底盘菌株,研究结果为通过批量敲除策略筛选新型致病因子奠定重要基础。     

果生刺盘孢效应蛋白CfEC92靶向Mal d 1j抑制苹果免疫

由果生刺盘孢引起的炭疽叶枯病是我国苹果产区的重要病害。果生刺盘孢在侵染苹果过程中分泌效应蛋白CfEC92促进其侵染,但其作用机制仍不清楚。本研究从苹果cDNA中克隆出Mal d 1j蛋白,结构域及氨基酸序列分析显示,Mal d 1j为PR10家族成员。Mal d 1j基因过表达能显著增强苹果对炭疽叶枯病抗性,而沉默Mal d 1j显著降低其抗性。在烟草中过表达Mal d 1j提高烟草抗性,共表达CfEC92和Mal d 1j蛋白,降低Mal d 1j对疫霉菌抗性。通过酵母双杂交、BIFC和Co-IP分析证明CfEC92与Mal d 1j可以发生直接互作,且其互作定位于细胞膜和细胞核,表明CfEC92 通过与Mal d 1j互作影响植物免疫。本研究揭示了果生刺盘孢效应蛋白CfEC92通过靶向Mal d 1j抑制植物免疫促进其侵染的分子机制,为苹果炭疽叶枯病防控提供了新的思路。     

三角梅苞叶色彩参数和色素含量分析及蓝色转基因受体的筛选北大核心CSCD

三角梅作为重要的观赏植物颜色繁多,但缺乏稀有的蓝色。为筛选适合的蓝色转基因受体,明确不同品种三角梅苞片吸收利用DHM(二氢杨梅素)合成甜菜色素途径竞争产物(类黄酮色素)的潜在能力,该研究对红色、白色、黄色和紫色4大花色6个品种的三角梅苞片进行离体诱导培养,测定诱导培养后苞片色彩参数及色素含量变化,并进行相关性分析。结果显示:(1)三角梅苞片红绿色相值(a*)是决定苞片呈色的主要色彩参数,其色彩主要由甜菜色素和黄酮类色素决定,并以甜菜红素的影响最大。(2)除白色品种三角梅苞片中黄酮类色素含量大于甜菜色素含量外,其余品种苞片发育中甜菜色素含量均呈上升趋势,黄酮类色素呈下降趋势。(3)甜菜色苷含量与苞片a*值呈显著正相关关系,同时与苞片黄蓝色相值(b*)呈显著负相关关系;总黄酮含量与苞片b*值呈显著正相关关系,与苞片a*值呈极显著负相关关系。(4)经DHM体外诱导培养后,苞片总黄酮含量及占比在4个品种三角梅(‘新加坡大白’、‘宝老橙’、‘中国丽人’、‘黄蝶’)中明显升高,但各品种苞片总甜菜色素含量及占比均下降,并以‘新加坡大白’苞片中总黄酮含量上升幅度最大(65.77%),含量占比变化(增加26.91%)也为6个品种中最大。(5)灰色关联度综合分析显示,白色品种‘新加坡大白’与灰色关联度分析拟定的参考品种关联度最高(0.7444),表明三角梅品种中‘新加坡大白’可考虑作为蓝色转基因三角梅的受体品种。     

拟南芥微丝加帽蛋白β亚基参与壳梭孢素诱导的气孔开放过程

气孔是植物响应外源信号,与环境进行水分和气体交换的门户。由外源信号引起的保卫细胞微丝骨架动态变化在气孔运动中发挥重要作用,但是具体的精确调节机制仍不清楚。微丝结合蛋白家族(ABPs) 是微丝动态组装最直接的调控者,它们的作用不容忽视。本文运用反向遗传学,以微丝结合蛋白—加帽蛋白 (CP) β-亚基 (CPB) 突变体cpb-3为实验材料,探究其在壳梭孢素 (FC)诱导气孔开放中的作用。结果发现:离体叶片干燥3 h,cpb-3突变体的叶片失水率为63.45%,明显高于野生型的48.99%。气孔开度测量及激光共聚焦显微镜观察发现,cpb-3突变体的气孔开放程度以及微丝动态重排对FC分子更敏感。气孔开度相比野生型增大了20% (P<0.05),含辐射状微丝排布的保卫细胞数量比例增幅达到58.3%,比对照组高出18.5%。此外,非损伤微测技术记录保卫细胞Ca²⁺、K⁺等跨膜运输动态,FC处理下,cpb-3突变体保卫细胞中Ca²⁺外流速度升至212.86 pmol cm^-2s^-1,野生型仅为68.76 pmol cm^-2s^-1,明显快于野生型。且K⁺内流也有相同表现。综上表明,微丝加帽蛋白CP的β亚基CPB可能通过调节保卫细胞微丝骨架动态重排以及离子流动,在FC诱导的气孔运动中发挥重要的作用。     

Homoplasy or plesiomorphy? Reconstruction of the evolutionary history of mitochondrial gene order rearrangements in the subphylum Neodermata

Recent mitogenomic studies have exposed a gene order (GO) shared by two classes, four orders and 31 species (‘common GO’) within the flatworm subphylum Neodermata. There are two possible hypotheses for this phenomenon: convergent evolution (homoplasy) or shared ancestry (plesiomorphy). To test those, we conducted a meta-analysis on all available mitogenomes to infer the evolutionary history of GO in Neodermata. To improve the resolution, we added a newly sequenced mitogenome that exhibited the common GO, Euryhaliotrema johni (Ancyrocephalinae), to the dataset. Phylogenetic analyses conducted on two datasets (nucleotides of all 36 genes and amino acid sequences of 12 protein coding genes) and four algorithms (MrBayes, RAxML, IQ-TREE and PhyloBayes) produced topology instability towards the tips, so ancestral GO reconstructions were conducted using TreeREx and MLGO programs using all eight obtained topologies, plus three unique topologies from previous studies. The results consistently supported the second hypothesis, resolving the common GO as a plesiomorphic ancestral GO for Neodermata, Cestoda, Monopisthocotylea, Cestoda + Trematoda and Cestoda + Trematoda + Monopisthocotylea. This allowed us to trace the evolutionary GO scenarios from each common ancestor to its descendants amongst the Monogenea and Cestoda classes, and propose that the common GO was most likely retained throughout all of the common ancestors, leading to the extant species possessing the common GO. Neodermatan phylogeny inferred from GOs was largely incongruent with all 11 topologies described above, but it did support the mitogenomic dataset in resolving Polyopisthocotylea as the earliest neodermatan branch. Although highly derived GOs might be of some use in resolving isolated taxonomic and phylogenetic uncertainties, we conclude that, due to the discontinuous nature of their evolution, they tend to produce artefactual phylogenetic relationships, which makes them unsuitable for phylogenetic reconstruction in Neodermata. Wider and denser sampling of neodermatan mitogenomic sequences will be needed to infer the evolutionary pathways leading to the observed diversity of GOs with confidence.     

AIM2 promotes non-small-cell lung cancer cell growth through inflammasome-dependent pathway

The human absent in melanoma 2 (AIM2) is considered as a DNA recognizer. AIM2 has been described as a tumor suppressor gene in the early years. But recent studies suggested that it functions as an oncogene in several cancers. However, its roles in non-small-cell lung cancer (NSCLC) remain unclear. Here we reported that AIM2 highly expressed in NSCLC cells and exhibited a tumor-promoting property both in vitro and in vivo. Besides, AIM2 short hairpin RNA (shRNA)-mediated suppression of cell proliferation was triggered by the accumulation of cells at the G2/M phase. Knockdown of AIM2 reduced the inflammasome formation, while overexpression of AIM2 or stimulation by poly(dA:dT) induced the inflammasome formation. Interestingly, blockade of the inflammasome by caspase-1 inhibitor VX-765 or ASC small interfering RNA (siRNA) abolished the effects brought by AIM2 shRNA and AIM2 plasmid. In summary, our results revealed that AIM2 functioned as an oncogene in NSCLC in an inflammasome-dependent way.     

Expression profile of circular RNA in rat intimal hyperplasia and target gene prediction

Intimal hyperplasia is an important cause of stenosis or occlusion after vascular injury. Circular RNAs (circRNAs) are known to be related to various cardiovascular diseases. However, the expression profile of circRNAs in the neointima has not been reported in detail. In this study, we established a rat common carotid artery (CCA) injury model. A microarray detection showed significant differences in circRNA expression between the normal and injured CCA. Real-time quantitative polymerase chain reaction verified the differences. We used bioinformatics to predict the microRNAs that possibly interact with the differentially expressed (DE) circRNAs and linked the potential functions of circRNAs to the target genes of the microRNAs. We believe that the DE circRNA in neointima may affect the differentiation, proliferation, and migration of vascular cells through a variety of target genes. The intervention or utilization of certain circRNAs should be a new method for preventing and treating intimal hyperplasia.     

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.     

Extracellular αB-crystallin modulates the inflammatory responses

Neuroinflammation is considered a challenging clinical problem. Chronic inflammatory responses play important roles in the onset and progression of various neurodegenerative diseases, including multiple sclerosis (MS). Previous studies have shown that astrocytes express small heat shock protein αB-crystallin (CRYAB) which is capable of inhibiting inflammatory responses in astrocytes per se. However, the underlying mechanisms of CRYAB-induced modulation of neuroinflammation are still not fully understood. In the present study, we investigated the role of extracellular CRYAB in the interaction between microglia and astrocytes in the context of MS-associated neuroinflammation. We found that the expression of CRYAB was profoundly increased in EAE mice. CRYAB was preferentially expressed in astrocytes and could be secreted via exosomes. Levels of exosomal CRYAB secreted from astrocytes were markedly increased under stress conditions. Furthermore, incubation of immortalized astrocytes or microglia cell lines with CRYAB remarkably suppressed astrocytes and microglia-mediated inflammatory responses in both autocrine and paracrine manners. Our results reveal a novel function for extracellular CRYAB in the regulation of neuroinflammation. Targeting extracellular CRYAB-modulated neuroinflammation is a potential therapeutic intervention for MS.     

Pre-colonization of PGPR triggers rhizosphere microbiota succession associated with crop yield enhancement

Plant and Soil - Plant growth-promoting rhizobacteria (PGPR) substantially improve plant growth and health, but their effects on the succession of rhizosphere microbiota throughout the growth...