过表达ZmIBH1-1提高玉米苗期抗旱性

【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T₃代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T₄代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。     

高效液相色谱法测定青贮饲料桑中的乳酸、乙酸和丙酸含量条件探讨

本文应用高效液相色谱法（HPLC）对青贮饲料桑中的乳酸、乙酸和丙酸含量测定进行了研究。通过对色谱柱、流动相、流速及样品处理条件进行优化，建立了一种应用HPLC法同时测定青贮饲料桑中乳酸、乙酸和丙酸含量的方法。研究结果表明：乳酸、乙酸和丙酸在一定浓度下具有良好的线性关系，且相关系数R2均大于0.999，加标回收率为98.35% ~ 104.24%，标准品中回收率和精密度试验相对标准偏差（RSD）为0.01%~0.58%，表明该方法准确性较好。3种物质检出限为4.928 ~ 9.489 mg/L，适用于青贮饲料桑中有机酸的定量检测。 [关键词] 高效液相色谱（HPLC）|青贮饲料桑|乳酸|乙酸|丙酸     

灵芝的生物学特性、液体发酵及药理作用的研究进展

以灵芝生长特性、液体发酵、药理作用为切入点,综合查询近5年发表的收录于中国知网和艾斯维尔等数据库中的相关文献,就灵芝的生物学特性、液体发酵条件、药理作用方面的研究进展进行综述,为更好地开发利用灵芝资源提供依据。指出现今灵芝的药理作用等基础研究较多,而生长栽培和发酵条件优化的研究报道则较少,而其临床应用的机制则有待进一步探讨。     

A rapid,simple, and sensitive immunoagglutination assay with silica nanoparticles for serotype identification of Pseudomonas aeruginosa

An agglutination test based on colored silica nanoparticles (colored SiNps) was established to detect serotypes of Pseudomonas aeruginosa. Monodisperse colored SiNps were used as agglutination test carriers. The colored SiNps were prepared through reverse microemulsion with reactive dyes, sensitized with 11 kinds of mono-specific antibodies against P. aeruginosa, and denoted as IgG-colored SiNps. Eleven kinds of IgG-colored SiNps were individually mixed with P. aeruginosa on a glass slide. Different serotypes of P. aeruginosa could be identified by agglutination test with evident agglutination. The P. aeruginosa could be detected in a range from 3.6 × 10⁵ to 3.6 × 10¹² cfu mL^?1. This new agglutination test was confirmed to be a specific, sensitive, fast, easy-to-perform, and cost-efficient tool for the routine diagnosis of P. aeruginosa.     

船舶噪声声压级对大黄鱼幼鱼游泳、摄食行为及免疫生理指标的影响

通过录制大黄鱼(Larimichthys crocea)产卵场附近船舶航行时的噪声,并以此为刺激源,以大黄鱼幼鱼为实验对象,研究了船舶噪声声压级对大黄鱼幼鱼游泳、摄食行为及免疫生理指标的影响。研究发现,当噪声声压级<60 dB时,幼鱼趋避游泳行为不强烈;但随着声压级增大,开始呈现出不同强度的趋避行为,依次表现出:游泳速度加快、鱼与鱼之间及鱼与桶壁之间发生碰撞、瞬间反应无序、跳跃等行为;当声压级>200 dB时,刺激2 min后就出现了死亡个体。在120 dB和150 dB短期单次和多次刺激下,幼鱼血浆中的皮质醇、血糖、血红蛋白和乳酸这4个与应激相关的生理指标均显著上升,其中皮质醇、血糖和乳酸上升幅度尤为明显。另外,皮质醇单次刺激后即达到峰值,而多次刺激后反而较单次刺激有所下降;血糖、血红蛋白和乳酸则具有累加效益,多次刺激要高于单次刺激。在120 dB长期(30 d)刺激下,幼鱼生长明显减缓,血浆中部分免疫指标(免疫球蛋白M,干扰素-α,白介素-1β和肿瘤坏死因子-α)明显降低,肠道菌群也发生显著变化,突出表现为部分益生菌如芽孢杆菌、乳杆菌等相对丰度明显降低。摄食行为影响方面,研究发现幼鱼能准确识别噪声源的位置,并具有一定的记忆性。研究结果揭示了船舶噪声对大黄鱼幼鱼的危害,可为今后制定大黄鱼产卵场的保护措施提供数据支撑。     

添加物对木耳液体菌种老化的影响

以黑木耳品种‘黑威15’为试验材料,研究营养物质对木耳液体菌种生长及老化的影响,筛选抗老化及易老化培养基,研究其抗氧化酶活性、底物降解酶活性、抗氧化物质及膜质过氧化程度变化规律。结果表明,①与对照组相比,添加磷酸二氢钾及VB1可延缓菌丝老化,且促进菌丝生长。添加硫酸镁对菌丝老化有缓解作用,但抑制菌丝生长。添加过量氮源可促进菌丝生长且加速菌丝老化。②与对照及易老化培养基相比,抗老化培养基中菌丝总蛋白含量、过氧化物酶、超氧化物歧化酶、抗坏血酸过氧化物酶、淀粉酶活性显著提高。表明活性氧自由基清除系统相关酶活性的提高对延缓菌丝老化具有重要作用。③SOD活性随菌龄增加而逐渐减小,与菌龄呈显著负相关。在抗老化培养基中,丙二醛含量随菌龄增加而降低,两者间呈显著负相关。在对照及易老化培养基中,MDA含量随菌龄增加呈上升趋势,两者间呈显著正相关。研究对分析木耳菌丝老化机制及解决液体菌种老化问题具有理论价值。     

不同作物秸秆腐解液对节节麦种子萌发和幼苗生长的影响

节节麦(Aegilops tauschii Coss.)作为麦田恶性杂草之一,给中国小麦生产带来了严重的经济损失。为探究农作物秸秆在节节麦生态防治方面的作用,本试验采用了滤纸培养皿法和小杯法研究了不同浓度的玉米、油菜、水稻秸秆及稻壳腐解液对节节麦种子萌发率、发芽指数及幼苗根长、苗高和干重5项生物指标的影响。结果表明,当浓度为6.25 mg/mL时,玉米、油菜秸秆腐解液对节节麦幼苗干重表现为促进作用,对其他测定指标均为抑制作用。稻壳腐解液除了对根长具有抑制作用外,对其他指标均具有促进作用。水稻秸秆腐解液对5项指标均具有促进作用。当浓度大于50 mg/mL时,4种秸秆腐解液对5项指标均具有抑制作用,且随着浓度的增加,抑制作用增强。总体来说,作物秸秆腐解液对节节麦的化感作用强弱依次为玉米秸秆、油菜秸秆、稻壳、水稻秸秆,且对根的化感作用尤为明显。     

Identification of an H1N1 subtype of swine influenza virus and serological analysis

To investigate the epizootic of swine influenza virus(SIV), 60 nasal swabs were collected from a clinical cases of pig farm in Tai'an City, Shandong Province of China in April 2017. SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced. An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1). Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch, seven genome segments belonged to avian-like H1N1 influenza virus lineage. The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G, which is a typical molecular biological characteristic. Five potential N-glycosylation sites(N14, N26, N277, N484 and N543) were found in the HA gene. To further investigate the epidemiology of SIV in this farm, the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens. The results showed that the total positive rate was 65.43%. The positive rates of single virus infection detected by EAH1N1, 2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35, 30.85 and 7.47%, respectively. The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm. These data provide evidence of an epizootic of SIV.     

Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.