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。     

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.     

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.     

大豆PAL2-3基因的克隆及转化研究

大豆异黄酮是苯丙氨酸代谢途径中合成的一类次级代谢产物,在苯丙氨酸代谢途径中,苯丙氨酸解氨酶(PAL)是关键酶和限速酶。本课题组前期研究发现PAL基因的相对表达量与大豆异黄酮含量具有明显的协同增减趋势,并且在PAL基因家族成员时空表达模式分析中发现,PAL2-3(XM_003542493)是PAL基因家族中相对表达量较高的主要表达成员之一。本试验首先通过克隆大豆中PAL2-3基因;然后构建pCAMBIA3301-GmPAL2-3植物过表达载体,将构建好的pCAMBIA3301-GmPAL2-3表达载体重组质粒转化到根癌农杆菌EHA105中;采用农杆菌介导的大豆子叶节转化体系获得转化植株,并对T1代转化植株进行PPT检测,外源标记基因Bar检测以及荧光定量PCR检测,对转基因阳性植株进行大豆籽粒异黄酮含量的测定。结果表明T_1代转基因植株中PAL2-3基因的表达量是对照的5.11~11.24倍,总异黄酮含量最高的(2 587.63μg·g~(-1))是对照(1 616.90μg·g~(-1))的1.6倍。因此在大豆中过量表达PAL2-3基因可以提高大豆籽粒中异黄酮含量。     

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.     

Detection of seven phytohormones in peanut tissues by ultra-highperformance liquid chromatography-triple quadrupole tandem mass spectrometry

Development of highly sensitive and reliable method for detection of phytohormones is of great significance to study plant hormones and agricultural production.In this study,an ultra-high-performance liquid chromatography-mass spectrometry/mass spectrometry method was established for separation and quantification of trans-zeatin,trans-zeatin riboside,gibberellin A_3,indol-3-acetic acid,salicylic acid,abscisic acid,and jasmonic acid(JA) without any label.The sepa ration was performed on an Agilent Explus Plus C18 column by using methanol and water as mobile phases with gradient elution.The target compounds were confirmed and quantified by mass spectrum via positive electrospray ionization for trans-zeatin,transzeatin riboside,indole-3-acetic acid,and via negative electrospray ionization for gibberellin3,salicylic acid,abscisic acid,and JA.The limits of detection ranged from 0.0127 ng L~(-1) for gibberellin A_3(GA_3) to 33.26 ng L~(-1) for JA and were lower than the currently reported values in literature.The proposed method was applied for qualitative and quantitative analyses of phytohormones in peanut gynophores and pods.The recoveries of the spiked phytohormones ranged from 80.20 to102.56%.The contents of seven endogenous hormones varied specifically in different development stages of peanuts.This study provides a highly sensitive and selective detection method for hormones and elucidates the growth and development of the gynophore and peanut fruit,which are controlled by seven endogenous hormones.     

玉米粉中伏马毒素FB1基体标准物质的评价

为填补我国在伏马毒素基体标准物质的空白,本试验研制了以玉米粉为基体的伏马毒素FB1标准物质,玉米样品经过筛、加标、冷冻干燥、磨粉、混匀、密封分装后,用超高效液相色谱-串联质谱(UPLC-MS/MS)法检验样品的均匀性、稳定性,幵联合多家实验室对玉米粉中伏马毒素FB1含量定值,同时分析样品的不确定度。样品均匀性经斱差分析法表明F值为1.42,小于临界值F_(0.05),且伏马毒素FB1含量在觃定时间6个月内无明显变化。结果表明,均匀性与稳定性均符合标准物质的要求。样品定值为1475.56μg kg~(–1),不确定度为169.98μg kg~(–1)。该标准物质可替代进口标准物质,用于伏马毒素检测过程中的仪器校准、实验室质量控制和操作人员的水平考核等。     

用液相色谱串联质谱法测定渔业水样中6种农药的含量

本文建立了一种快速检测渔业水质样本中酰胺类除草剂乙草胺、丙草胺、丁草胺、三嗪类除草剂莠去津、烟碱类杀虫剂噻虫嗪、三唑类杀菌剂苯醚甲环唑6种农药含量的液相色谱串联质谱法。采用乙腈-水-甲酸梯度洗脱、电喷雾电离方式、多反应监测模式测定考察检出限、线性范围、日内精密度及日间精密度。在三种加标浓度下,该方法的回收率为78.2%~104.7%。用液相色谱-四级杆-飞行时间质谱对疑似目标物质进行定性,用精确质量数、同位素丰度比和二级碎片解析方式等解析定性结构。该方法能够满足检测这六种农药的要求,能准确定性,自建的谱库可为渔业污染事故鉴定和水产品质量安全风险评估做支撑。     

5种动物源食品中的氟虫腈及其3个代谢物残留量检测

利用QuEChERS技术结合超高效液相色谱-串联质谱的方法检测5种动物源食品(鸡肉、鸡蛋、猪肝、牛奶、猪肉)中氟虫腈及其3个代谢物残留量。样品用乙腈提取,经低温处理,多壁碳纳米管(MWCNTs)、弗罗里硅土(Florisil)和C18分散固相萃取(d-SPE)净化,外标法定量。在不同浓度的加标水平下,氟虫腈及其代谢物在5种动物源食品中的平均回收率为72.1%～113.8%,相对标准偏差RSD为1.9%～17.6%,检出限LOD在0.73～1.94μg/kg范围内,定量限LOQ在5～10μg/kg范围内。该方法具有简单、灵敏、准确等优点,适用于动物源食品中氟虫腈及其代谢物的快速筛查和定量检测。