枯草芽孢杆菌聚谷氨酸合成途径相关基因功能研究

聚谷氨酸(polyglutamic acid, PGA)作为一种天然多功能的聚合物,近年来成为研究的热点。由于很难通过化学方法合成,微生物发酵是目前生产聚谷氨酸的有效途径。【目的】从基因水平探究枯草芽孢杆菌聚谷氨酸合成途径中degS、degQ、degU、swrA、rocA、putM基因的功能,通过分子改造实现对代谢途径的调控。【方法】以枯草芽孢杆菌为出发菌株,通过对代谢途径中相关基因进行敲除或过表达,分别构建degS、degQ和degU基因缺失的重组菌,swrA、rocA和putM基因过表达的重组菌,借助菌株胞外聚谷氨酸积累的变化分析影响途径的关键节点。【结果】在摇瓶发酵条件下,重组菌Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA、Bacillus subtilis 168-putM的胞外聚谷氨酸含量分别是原始菌株的1.28倍、1.47倍和1.37倍。重组菌Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-ΔdegU的胞外...     

生物有机肥对菠萝心腐病及根际土壤细菌群落的影响

【目的】探明施用生物有机肥对菠萝心腐病的防控效果及对根际土壤微生物的影响。【方法】本研究采用高通量测序技术,综合分析不同施肥措施根际土壤微生物细菌群落多样性及群落特征。【结果】相比常规施肥处理(CK)和普通有机肥处理(YJ),生物有机肥处理KN (羊粪有机肥+泥炭土+枯草芽孢杆菌)和生物有机肥处理KY (羊粪有机肥+椰糠+枯草芽孢杆菌)均能显著降低菠萝心腐病的发病率,且KN处理的防控效果最佳。生物有机肥(KN、KY)施入后土壤细菌α多样性指数高于CK和YJ处理,并形成了明显不同的细菌群落结构。与CK相比,生物有机肥KN处理显著提高了拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)的丰度,KY处理中的酸杆菌门(Acidobacteria)、绿弯菌门(Chloroflexi)、芽单胞菌门(Gemmatimonadetes)、硝化螺旋菌门(Nitrospirae)丰度显著增加;属水平上,生物有机肥中的蔗糖伯克霍尔德菌属(Paraburkholderia)和黄杆菌属(Flavobacterium)丰度均显著提升。方差分区分析(variance partitioning analysis, VPA)表明,土壤化学性质(36.29%)对细菌群落影响最大,其中土壤速效钾和有机质是影响土壤细菌群落的关键因子,此外发病率(22.53%)和肥料偏生产力(16.42%)也是影响土壤细菌群落的重要因子。【结论】施用生物有机肥(KN、KY)能改变根际土壤细菌群落结构,降低发病率,对菠萝根际土壤生态系统稳定与健康具有重要意义。     

人体肠道细菌寡营养培养组条件的优化研究

【目的】探讨寡营养对人体肠道细菌培养组的条件。【方法】通过稀释富集培养基、固体平板和增菌肉汤培养基成分获得寡营养培养基。对健康人粪便样本分别用原液(0)、5、10、20、30和40倍稀释的富集培养基(添加羊血和瘤胃液的血培养瓶)连续增菌,在不同时间点(第0、3、6、9、15、27、30天)吸取增菌液,用YCFA (yeast casitone fatty acid)固体培养平板分离菌落;用YCFA增菌肉汤增菌后再次挑取单菌落,利用基质辅助激光解吸/电离飞行时间(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF)质谱和16S rRNA基因测序鉴定菌株。通过比较上述6种寡营养条件分离肠道菌群的效果,选取富集培养基原液、稀释10倍和30倍这3 种条件下分离效果较好的富集条件,与同样稀释倍数条件的固体平板和增菌肉汤分别组合成9种培养基条件,进一步优化肠道菌群的培养组条件。【结果】在6种寡营养富集培养基中,未稀释(原液)、10 倍和30倍稀释的富集培养基分离细菌的种类比其他...     

棒状腐败乳杆菌Lc7的生物学特性及益生作用

【目的】对分离自健康成人粪便样本的棒状腐败乳杆菌(Loigolactobacillus coryniformis)Lc7进行分类学鉴定和益生潜力评估。【方法】基于16SrRNA基因和基因组核心基因构建系统发育树,对Lc7进行分类学鉴定;通过耐酸和胆汁酸盐、粘附、抗氧化和抑菌实验,以及溶血、明胶酶活性和抗菌药物敏感性实验,评估Lc7的益生特性。同时,构建小鼠溃疡性结肠炎模型,评估Lc7的体内抗炎潜力。【结果】Lc7鉴定为L. coryniformis,在酸和胆汁酸盐的连续作用下,Lc7的存活率为70.17%。Lc7对HT-29细胞的粘附指数为56.33 CFU/cell,其自聚集和疏水性分别为80%和40%;Lc7对福氏志贺菌和鼠伤寒沙门菌等7个常见致病菌均有较强的抑制能力;对1,1-二苯基-2-苦基肼(1,1-diphenyl-2-picryl-hydrazyl,DPPH)和羟自由基(hydroxyl radicals,·OH)的清除率分别为91.70%和48.53%;Lc7无溶血现象和明胶酶活性,对选取的大多数抗生素均敏感。在小鼠结肠炎实验中,Lc7干预组小鼠结肠长度明显长于模型组(...     

野芹菜体细胞胚胎发生早期变化的细胞学研究

通过光学与电子显微镜观察研究了野芹菜(Angelica polymorpha Maxim.)叶柄外植体胚性细胞的起源与原胚状体的发生。叶柄切段植入 MS 2mg/L2,4-D 0.25 mg/L KT固体培养基后,以DNA合成和细胞分裂为指标,判明了胚状体发生与紧贴维管束的鞘细胞层密切相关;鞘细胞通过有丝分裂形成多层结构的细胞群,它们仍含大液泡及薄层胞质,成片被覆在维管束表面;胚性细胞团即不同步地发生在该多层组织较内层的局部位点上,细胞具大核,胞质稠密,经持续有丝分裂发展成大小不同呈瘤状突起的原胚状体。伴随着鞘细胞的剧烈变化,韧皮部薄壁细胞亦分裂增殖,原有筛管变形衰退,部分新增殖的细胞分化为新筛分子;木质部中,原导管束附近朝向韧皮部一侧的部分薄壁细胞亦进一步分化为孔纹导管。外植体中维管系统的再次分化,显然是与输导功能强化以适应原胚状体发生时对营养物质的大量需求有关。     

高耐酸酒酒球菌常压室温等离子体诱变选育及其苹果酸-乳酸发酵研究

【目的】为选育出高度耐酸性酒酒球菌（Oenococcus oeni）突变菌株,研究其胁迫耐受性能及苹果酸-乳酸发酵（malolactic fermentation,MLF）能力。【方法】以酒酒球菌SD-2a为出发菌株,通过常压室温等离子体（atmospheric and room temperature plasma,ARTP）诱变技术,筛选高耐酸性酒酒球菌突变菌株,并探究其乙醇耐受性及在模拟酒和葡萄酒条件下的MLF能力。【结果】经过ARTP诱变处理后,利用pH 3.0的胁迫传代培养和分离纯化等,获得了5株β-葡萄糖苷酶活性较好的耐酸突变菌株,且在高乙醇浓度下表现出了较好的耐乙醇性。其中突变菌株ARTP-2在模拟酒中的β-葡萄糖苷酶活性和l-苹果酸累积降解量最高,且其在葡萄酒中l-苹果酸降解速率快于出发菌株,在第18天完成MLF,发酵后的葡萄酒香气成分的含量显著高于接种SD-2a的酒样。【结论】突变菌株ARTP-2具有良好的胁迫耐受性和MLF能力,对葡萄酒的香气起到积极的作用,为进一步开发优质的MLF商业发酵剂奠定基础。     

The complex genome and adaptive evolution of polyploid Chinese pepper (Zanthoxylum armatum and Zanthoxylum bungeanum)

Zanthoxylum armatum and Zanthoxylum bungeanum, known as ‘Chinese pepper’, are distinguished by their extraordinary complex genomes, phenotypic innovation of adaptive evolution and species-special metabolites. Here, we report reference-grade genomes of Z. armatum and Z. bungeanum. Using high coverage sequence data and comprehensive assembly strategies, we derived 66 pseudochromosomes comprising 33 homologous phased groups of two subgenomes, including autotetraploid Z. armatum. The genomic rearrangements and two whole-genome duplications created large (~4.5 Gb) complex genomes with a high ratio of repetitive sequences (>82%) and high chromosome number (2n = 4x = 132). Further analysis of the high-quality genomes shed lights on the genomic basis of involutional reproduction, allomones biosynthesis and adaptive evolution in Chinese pepper, revealing a high consistent relationship between genomic evolution, environmental factors and phenotypic innovation. Our study provides genomic resources and new insights for investigating diversification and phenotypic innovation in Chinese pepper, with broader implications for the protection of plants under severe environmental changes.     

Upgrading the genome of an elite japonica rice variety Kongyu 131 for lodging resistance improvement

Developing a new rice variety requires tremendous efforts and years of input. To improve the defect traits of the excellent varieties becomes more cost and time efficient than breeding a completely new variety. Kongyu 131 is a high-performing japonica variety with early maturity, high yield, wide adaptability and cold resistance, but the poor-lodging resistance hinders the industrial production of Kongyu 131 in the Northeastern China. In this study, we attempted to improve the lodging resistance of Kongyu 131 from perspectives of both gene and trait. On the one hand, by QTL analysis and fine mapping we discovered the candidate gene loci. The following CRISPR/Cas9 and transgenic complementation study confirmed that Sd1 dominated the lodging resistance and favourable allele was mined for precise introduction and improvement. On the other hand, the Sd1 allelic variant was identified in Kongyu 131 by sequence alignment, then introduced another excellent allelic variation by backcrossing. Then, the two new resulting Kongyu 131 went through the field evaluation under different environments, planting densities and nitrogen fertilizer conditions. The results showed that the plant height of upgraded Kongyu 131 was 17%–26% lower than Kongyu 131 without penalty in yield. This study demonstrated a precise and targeted way to update the rice genome and upgrade the elite rice varieties by improving only a few gene defects from the perspective of breeding.     

TaTPP-7A positively feedback regulates grain filling and wheat grain yield through T6P-SnRK1 signalling pathway and sugar–ABA interaction

Grain size and filling are two key determinants of grain thousand-kernel weight (TKW) and crop yield, therefore they have undergone strong selection since cereal was domesticated. Genetic dissection of the two traits will improve yield potential in crops. A quantitative trait locus significantly associated with wheat grain TKW was detected on chromosome 7AS flanked by a simple sequence repeat marker of Wmc17 in Chinese wheat 262 mini-core collection by genome-wide association study. Combined with the bulked segregant RNA-sequencing (BSR-seq) analysis of an F₂ genetic segregation population with extremely different TKW traits, a candidate trehalose-6-phosphate phosphatase gene located at 135.0 Mb (CS V1.0), designated as TaTPP-7A, was identified. This gene was specifically expressed in developing grains and strongly influenced grain filling and size. Overexpression (OE) of TaTPP-7A in wheat enhanced grain TKW and wheat yield greatly. Detailed analysis revealed that OE of TaTPP-7A significantly increased the expression levels of starch synthesis- and senescence-related genes involved in abscisic acid (ABA) and ethylene pathways. Moreover, most of the sucrose metabolism and starch regulation-related genes were potentially regulated by SnRK1. In addition, TaTPP-7A is a crucial domestication- and breeding-targeted gene and it feedback regulates sucrose lysis, flux, and utilization in the grain endosperm mainly through the T6P-SnRK1 pathway and sugar–ABA interaction. Thus, we confirmed the T6P signalling pathway as the central regulatory system for sucrose allocation and source–sink interactions in wheat grains and propose that the trehalose pathway components have great potential to increase yields in cereal crops.