甲烷代谢古菌分离培养研究进展

全球变暖是全人类面临的一个巨大挑战,而温室气体排放持续上升是全球变暖的关键因素,并引发一系列生态环境问题。甲烷是第二温室气体,对全球变暖的贡献达20%。然而,在甲烷代谢中发挥重要作用的产甲烷古菌和厌氧甲烷氧化古菌(anaerobic methanotroph,ANME)较难培养,极大地限制了人们对甲烷代谢及其影响碳源-汇关系与机制的研究。本文综述了最新产甲烷古菌和ANME富集、分离和培养方法,包括富集培养、原位培养、共培养、微流控技术、稀释分离和固体分离技术、ANME反应器和培养瓶富集培养,以及宏基因组预测和反向基因组学,并对这些方法的优缺点进行了评估,对未来甲烷代谢古菌的富集、分离和培养提出新的建议。     

炼锌废渣-修复植物-凋落物体系的生态化学计量学研究

为揭示金属冶炼废渣堆场生态修复多年后,废渣-植物-凋落物系统中养分循环和系统维持机制。该研究以实现生态修复6 a的黔西北铅锌冶炼废渣堆场上土荆芥(Dysphania ambrosioides)、芦竹(Arundo donax)、刺槐(Robinia pseudoacacia)、构树(Broussonetia papyrifera)和柳杉(Cryptomeria fortunei)五种优势修复植物为对象,分析它们的主要营养器官(细根、粗根、茎/干、枝、叶片)、地表凋落物、植被下方表层废渣(0～10 cm)中碳(C)、氮(N)、磷(P)含量及化学计量特征,探讨它们之间的相互关系。结果表明:不同植物、不同营养器官间C、N、P的含量具有显著差异(P<0.05),C平均含量在两种草本植物中为茎>叶片>根>凋落物,在三种乔木中为干>枝>细根>粗根>叶片>凋落物; N和P的分布在草本植物中分别为叶片>凋落物>根>茎和叶片>根>凋落物>茎,在三种乔木中均为叶片>细根>凋落物>粗根>枝>...     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

湘南某矿区蔬菜中Pb、Cd污染状况及健康风险评估

通过采集湘南某矿区周边农田中种植的蔬菜和对应耕作层土壤样品,探究了农田蔬菜Pb、Cd的污染状况,蔬菜和土壤重金属含量之间的相关性,蔬菜中重金属对人体的健康风险。结果表明:(1)污染区土壤重金属污染非常严重,叶菜类蔬菜重金属超出了食品卫生标准限值,但是果菜类蔬菜中重金属没有超出食品卫生标准限值。(2)不同蔬菜不同器官所含Pb、Cd有明显差异;不同种类蔬菜Pb、Cd含量不同,一般是叶菜类果菜类;同种蔬菜不同部位重金属含量的顺序也不同,一般为根茎叶果,或根叶茎果。(3)二类蔬菜中重金属Pb、Cd的富集系数顺序为叶菜类果菜类。(4)蔬菜中Pb、Cd含量与土壤中相对应元素含量均无显著相关性。(5)就农产品安全性而言,果菜类蔬菜比叶菜类蔬菜更适合在此矿区栽培。     

SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.     

滇龙胆与青叶胆环烯醚萜类物质计量特征分析?

龙胆科龙胆属滇龙胆( Gentiana rigescens)与獐牙菜属青叶胆( Swertia mileensis)为我国特有种。采用高效液相色谱法测定其中马钱苷酸、獐牙菜苦苷、龙胆苦苷和当药苷含量,结合多变量分析研究环烯醚萜类成分含量及其构成比例在龙胆科植物种内和种间的差异。研究结果显示,4种环烯醚萜类成分的计量特征呈现种间差异,依据环烯醚萜类成分含量及其构成比例,所有滇龙胆样品可分为4类。马钱苷酸含量与纬度呈极显著负相关( R＝－0?348, P<0?05),与海拔呈极显著正相关( R＝0?307, P<0?01);獐牙菜苦苷含量与经度呈极显著负相关( R＝－0?592, P<0?01),高海拔有利于植株中当药苷构成比例的增加( R＝0?245, P<0?05);地理因子对植株中龙胆苦苷含量变化影响不显著( P>0?05)。     

Gibberellic acid is selectively downregulated in response to aphid-induced gall formation

The fluid-feeding aphid Schlechtendalia chinensis (Bell) induces horned galls on its primary host, the Chinese Sumac (Rhus chinensis Mill). Horned galls are harvested for their high content of tannins, and used in a range of medical and chemical applications. Gall development is a complex and highly controlled physiological process, where the growing insect population manipulates the plant developmental programs that allow the transformation of plant tissue into a gall. In this study, we examine whether Schlechtendalia alters the balance of plant hormones in the host tree as a means to achieve gall formation. For this, we measured concentrations for a series of endogenous hormones, including indole-3-acetic acid (IAA), cytokinin (CTK), gibberellic acid (GA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ETH). Specifically, we conducted a time course (namely, 30, 85, 100, 115, 125, 140, 155, and 170 days from gall initiation) analysis, where we measured both gall and leaf samples representing different developmental stages that spanned an entire growing season. To correlate these hormone data with developmental parameters during gall growth, we determined gall volume, tannin content, and aphid population size for the same time points. Interestingly, tannin production rose steeply in the early stages of gall development, while the aphid population size grew little. After this single peak (day 100), tannin concentrations declined moderately and aphid population size increased from then on. This switch in population growth was accompanied by notable changes in plant hormone titers. In general, all hormones but GA were elevated in all sample types isolated from the host tree (gall, leaves near and distant from gall) when compared with samples from an uninfected tree. Most of the elevated hormones showed similar changes over time; however, GA appeared to display the opposite behavior in all samples, suggesting that GA is a key target for controlling gall growth. When tannin concentrations spiked, GA levels peaked as well, while the remaining plant hormones exhibited a decline at that time. Principle component analysis revealed distinct functional groups in our hormone cohort. This yielded three groups comprising (1) CTK, ABA, ETH, and JA, (2) IAA and SA, (3) GA. The fact that GA comprised its own group and exhibited a unique profile during gall development prompted us to examine whether exogenous GA would alter the rate of gall growth. Indeed, we found that ectopic GA significantly accelerated gall growth, and more strongly than all other hormones, consistent with the notion that controlling GA levels within the gall is crucial for stimulating gall development. We propose a model, whereby the host plant downregulates GA concentrations in an attempt to throttle gall growth, while the gall-inducing aphid population counters these attempts.     

Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii)

Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T₁ mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.     

Synergistic inhibitory effects by the combination of gefitinib and genistein on NSCLC with acquired drug-resistance in vitro and in vivo

In clinical practice, most patients with non small cell lung cancer (NSCLC) who respond to tyrosine kinase inhibitors eventually progress because of an acquired resistance mutation, T790M, in epidermal growth factor receptor (EGFR). Thus, it is important to identify a new drug to reduce resistance. The aim of this study was to test whether genistein combined with gefitinib is effective against NSCLC in a cell line carrying T790M, and to clarify the underlying mechanisms. The human lung cancer cell line H1975 was used as an in vitro and in vivo model. Cells were treated with gefitinib, genistein, or a combination at a range of concentrations. Cell proliferation was calculated to assess the anticancer effects of the compounds in vitro. Flow cytometry and Western blotting were employed to determine the inhibitory effects on proliferation and the induction of apoptosis. The in vivo effects of the compounds were examined using a xenografted nude mouse model for validation. Gefitinib together with genistein enhanced both growth inhibition and apoptosis; however, the greatest synergistic effect was observed at low concentrations. p-EGFR, p-Akt, and p-mTOR expressions in vitro were reduced more by the combined use of the drugs, whereas caspase-3 and PARP activities were increased. Significantly more tumor growth inhibition was detected following combination treatment in the in vivo model. These findings suggest that genistein enhanced the antitumor effects of gefitinib in a NSCLC cell line carrying the T790M mutation. This synergistic activity may be due to increased inhibition of the downstream molecular and pro-apoptotic effects of EGFR.     

Membrane-bound pyrophosphatase of human gut microbe Clostridium methylpentosum confers improved salt tolerance in Escherichia coli,Saccharomyces cerevisiae and tobacco

Membrane-bound pyrophosphatases (PPases) are involved in the adaption of organisms to stress conditions, which was substantiated by numerous plant transgenic studies with H⁺-PPase yet devoid of any correlated evidences for other two subfamilies, Na⁺-PPase and Na⁺,H⁺-PPase. Herein, we demonstrate the gene cloning and functional evaluation of the membrane-bound PPase (CmPP) of the human gut microbe Clostridium methylpentosum. The CmPP gene encodes a single polypeptide of 699 amino acids that was predicted as a multi-spanning membrane and K⁺-dependent Na⁺,H⁺-PPase. Heterologous expression of CmPP could significantly enhance the salt tolerance of both Escherichia coli and Saccharomyces cerevisiae, and this effect in yeast could be fortified by N-terminal addition of a vacuole-targeting signal peptide from the H⁺-PPase of Trypanosoma cruzi. Furthermore, introduction of CmPP could remarkably improve the salt tolerance of tobacco, implying its potential use in constructing salt-resistant transgenic crops. Consequently, the possible mechanisms of CmPP to underlie salt tolerance are discussed.