桑树内生拮抗菌的分离鉴定及其对桑断枝烂叶病的生防初探

【目的】本研究从健康桑树茎中分离筛选对桑断枝烂叶病菌具显著拮抗作用的内生细菌,为该病生物防治奠定研究基础。【方法】采用组织培养法分离桑树内生菌,抑菌圈法和平板对峙法筛选抑菌活性稳定的内生拮抗菌;根据形态学、生理生化特征检测和基于16SrDNA、gyrA和gyrB基因的系统发育分析对拮抗菌进行菌种鉴定;利用抑菌圈法测定拮抗菌株活性发酵液热稳定性,菌丝生长速率法检测活性发酵液抑菌谱;并通过观察拮抗菌对桑断枝烂叶病菌BoeremiaexiguaGXH1菌株生长及菌丝形态的影响,扩增抑菌活性物质合成关键基因,以及采用酸沉淀法提取拮抗菌株脂肽类化合物并进行高效液相色谱串联质谱分析(LC-MS),初步探究可能的抑菌机制。【结果】从健康桑树茎中共分离获得17株桑树内生细菌,并从中筛选获得一株对桑断枝烂叶病菌B.exiguaGXH1有稳定拮抗作用的桑树内生细菌NPJ13菌株。该菌株形态学、生理生化特征与芽孢杆菌属一致,基于16SrDNA、gyrA和gyrB基因序列的系统发育分析结果显示该菌株与贝莱斯芽孢杆菌(Bacillusvelezensis)的亲缘关系最近,且处于系统发育树的最小分枝,故将NPJ13菌株鉴定为贝莱斯芽孢杆菌,命名为B. velezensis NPJ13。NPJ13菌株对灰霉病菌SWU5、核地杖菌SXSG-5、核盘菌PZ-2及烟草疫霉SWU20等12种病原真菌具有不同程度的拮抗作用,其活性发酵液具有较好的热稳定性。NPJ13菌株会导致桑断枝烂叶病菌GXH1菌丝发生扭曲、膨大、透明度增加、断裂等畸变现象;基因检测结果显示NPJ13菌株基因组中具有PKSI、NRPS、Sfp、ItuD、Srfc等5种抑菌活性物质合成关键基因,LC-MS检测结果表明菌株NPJ13脂肽类粗提物中含有表面活性素和伊枯草菌素。【结论】本研究分离筛选获得一株对桑断枝烂叶病菌具有显著拮抗作用的桑树内生细菌B. velezensis NPJ13菌株,为桑断枝烂叶病的生物防治提供了候选菌株。     

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰

[目的]DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。[方法]首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spfBCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了SpfB蛋白与DNA结合序列。[结果]spfB基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spfB基因的回补能够恢复该表型,证明spfB基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在△spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spfBCDE的启动子区域5''-TGTTTGT-3''相结合。[结论]SpfB作为转录调控因子负调控磷硫酰化修饰基因spfBCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

发酵饲料对生长育肥猪结肠微生物发酵及菌群组成的影响

【目的】研究复合菌发酵饲料对生长育肥猪结肠发酵、结肠黏膜与结肠内容物菌群组成的影响。【方法】采用气相色谱法检测育肥猪结肠内容物中挥发性脂肪酸浓度;采用MiSeq高通量测序方法检测育肥猪结肠黏膜与内容物中细菌菌群组成。【结果】饲喂发酵饲料对结肠黏膜及内容物中菌群多样性无显著影响(P0.05);显著提高了猪结肠黏膜中魏斯菌属和柔嫩梭菌属的相对丰度(P0.05),提高了结肠内容物中魏斯菌属、Subdoligranulum菌属相对丰度(P0.05);饲喂发酵饲料对结肠内容物中pH、乳酸、乙酸、丙酸、异丁酸、戊酸、异戊酸和总挥发性脂肪酸浓度无显著影响(P0.05),但显著提高了结肠内容物中的丁酸水平(P0.05)。【结论】饲喂复合菌发酵饲料可在一定程度上影响育肥猪结肠中细菌菌群的组成,促进丁酸生成,对肠道健康具有改善作用。     

Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).     

Alleviation of the detrimental effect of water deficit on wheat (Triticum aestivum L.) growth by an indole acetic acid-producing endophytic fungus

Plant and Soil - Endophytic fungi colonization is an eco-friendly strategy to respond to environmental stresses and confer tolerance to the host plant. Here, the responses of wheat plant inoculated...     

Genome‐wide association study identifies variation of glucosidase being linked to natural variation of the maximal quantum yield of photosystem II

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, F_v/F_m) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in F_v/F_m, which shows low level of variations in plants under non‐stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome‐wide association study (GWAS) and transgenic analysis in a rice mini‐core collection. We report here that four single‐nucleotide polymorphisms, located in the promoter region of β‐glucosidase 5 (BGlu‐5), are associated with observed variation in F_v/F_m. Indeed, our transgenic analysis showed a good correlation between BGlu‐5 and F_v/F_m. Thus, our work demonstrates the feasibility of using GWAS to study natural variation in F_v/F_m, suggesting that cis‐element polymorphism, affecting the BGlu‐5 expression level, may, indirectly, contribute to F_v/F_m variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.     

Physiological,Biochemical and Molecular Responses of Barley Seedlings to Aluminum Stress

Barley (Hordeum vulgare L.) is one of the most Aluminum (Al) sensitive cereal species. In this study, the physiological, biochemical, and molecular response of barley seedlings to Al treatment was examined to gain insight into Al response and tolerance mechanisms. The results showed that superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were inhibited to different degrees following Al exposure. The MDA content also significantly increased with increasing Al concentrations. SRAP results indicated significant differences between Al treatments and controls in terms of SRAP profile, and the genomic template stability (GTS) decreased with increasing Al concentration and duration. These integrative results help to elucidate the underlying mechanisms that the barley response to Al toxicity.     

Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here that Arabidopsis KEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although single kea4,5,6 mutants showed similar phenotype as the wild type under various conditions, kea4/5/6 triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺ and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that the kea4/5/6 triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺ condition. The cell wall‐derived pectin homogalacturonan (GalA)₃ partially suppressed the growth defects and ionic toxicity in the kea4/5/6 triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺ stress.