利用HPLC-ICP-MS方法测定中华绒螯蟹不同形态砷的残留量

采用高效液相色谱串联电感耦合等离子体质谱仪(HPLC-ICP-MS)测定中华绒螯蟹可食部位6种砷形态的残留量。经过前处理、提取和脱脂后,通过色谱柱、流动相、pH和流速等参数调节,筛选得到最优化的分离和检测条件。结果表明,6种砷形态的混合标准溶液在质量浓度0~50 μg/L范围时,均呈现良好的线性关系,其R²达到0.999,检测限为0.0060~0.0912 μg/kg,定量限为0.020~0.304 μg/kg。在样品中添加0.5、5、50 μg/L浓度水平,回收率良好,范围是70%~120%,相对标准偏差(RSD)均<5%,方法精密度与重复性均良好。本方法为中华绒螯蟹可食部位砷形态残留量建立了有效检测方法,为水产品砷残留快速检测提供技术支撑。     

粪肠球菌生长曲线的测定及其对小鼠脑组织的影响

为测定致羔羊脑炎粪肠球菌（Enterococcus faecalis）的生长曲线，寻求一种快速而准确的方法测定不同生长时期粪肠球菌数量，并客观评价其毒性强弱及其对小鼠脑组织的影响，试验采用平板菌落计数法和OD-Monitor振荡比浊法（D_λ值法）测定粪肠球菌的生长曲线，探究该菌在合适时间段内的吸光值（D_{600 nm}）与平板菌落计数法测定的活菌数（CFU）的关系。用粪肠球菌感染小鼠，观察记录小鼠的死亡情况，最后采用Karber法计算粪肠球菌感染小鼠的半数致死量（LD₅₀）。用LD₅₀的剂量感染小鼠，及时采集死亡小鼠脑组织，未死亡的小鼠72 h后全部剖杀取脑组织，一部分做涂片染色，制作病理切片，观察病理变化；一部分进行培养，用于PCR方法进行细菌的回收鉴定。结果显示，用两种方法测定此株粪肠球菌的生长曲线基本一致，在2~8 h生长迅速，为对数生长期，8~14 h生长缓慢，为稳定期，14 h之后死亡数增加，进入衰亡期；对12 h粪肠球菌D_{600 nm}与CFU的关系进行探讨，成功建立回归方程：y=20.769x-1.3422，R²=0.997；其感染小鼠的LD₅₀为7.77×10¹¹个活菌。以此剂量感染小鼠，脑组织涂片染色和培养染色，均能看到革兰氏阳性球菌；PCR结果显示，均出现了大小为112 bp的条带。对脑组织进行病理学观察发现该菌可导致脑组织充血、出血、形成微血栓，脑膜充血。通过生长曲线和其D_{600 nm}与CFU关系的建立，可实时监测粪肠球菌数量，为后期更深入研究粪肠球菌穿越血脑屏障的机制奠定重要的理论基础。     

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.     

Disulphide structure of high-molecular-weight (HMW-) gliadins as affected by terminators

This study aimed at elucidating SS-bonds of HMW-gliadins (HGL) from wheat with the focus on terminators of glutenin polymerisation. HGL from wheat flour extracts non-treated or treated with the S-alkylation reagent N-ethylmaleinimide (NEMI) were compared. HGL from wheat flour Akteur were isolated, hydrolysed with thermolysin and the resulting peptides pre-separated by gel permeation chromatography and analysed by liquid chromatography/mass-spectrometry using alternating electron transfer dissociation/collision-induced dissociation. Altogether, 22 and 28 SS-peptides from samples without and with NEMI treatment, respectively, were identified. Twenty-six peptides included standard SS-bonds of α- and γ-gliadins, high-molecular-weight and low-molecular-weight glutenin subunits. Eleven SS-bonds were identified for the first time. Fifteen peptides unique to HGL contained cysteine residues from gliadins with an odd number of cysteines (ω5-, α- and γ-gliadins). Thus, gliadins with an odd number of cysteines, glutathione and cysteine had acted as terminators of glutenin polymerisation. Decisive differences between samples without and with NEMI treatment were not obvious showing that the termination of polymerisation was already completed in the flour. The two HGL samples, however, were different in the majority of ten peptides that included disulphide-linked low-molecular-weight (LMW) thiols such as glutathione and cysteine with the former being enriched in the non-treated HGL-sample.     

茶树牻牛儿基牻牛儿基焦磷酸合成酶基因CsGGDPS的克隆及表达分析

以铁观音芽叶为材料,验证从茶树转录组数据库中筛选到的1条牻牛儿基牻牛儿基焦磷酸合成酶(GGDPS)的编码全长序列c DNA。该c DNA全长1 661 bp,含有1个1 137 bp完整的开放阅读框,命名为Cs GGDPS。该基因编码378个氨基酸,氨基酸序列具有类异戊二烯合成酶家族的5个保守域和2个特征功能域;序列分析显示,该c DNA与其他植物GGDPS高度保守,与三七(Panax notoginseng)的亲缘关系最近。Cs GGDPS属于不稳定、亲水蛋白,可能定位到叶绿体中,不存在跨膜结构,无信号肽,发生磷酸化的位点可能有20个;二级结构主要由α-螺旋构成,三级结构与拟南芥GGPPS11匹配度最高。实时荧光定量PCR结果表明,在茶树发育过程和不同叶位Cs GGDPS的表达量随芽叶成熟度的增加呈上升趋势,随着做青过程的进行,Cs GGDPS的表达量逐渐升高;Cs GGDPS在父本黄旦、母本铁观音和子一代金观音中均有表达,但表达量存在差异。     

茶树GGPS基因家族的克隆、生物信息学和表达分析

牻牛儿基牻牛儿基焦磷酸合酶(Geranylgeranyl diphosphate synthase,GGPS)是萜类合成途径的结构酶,对植物生长发育具有重要意义。本研究通过RACE和RT-PCR方法克隆得到5条潜在的茶树GGPS序列,分别命名为CsGGPS1-4和CsGGPS9,其中CsGGPS9存在3条等位基因,分别是CsGGPS9-1、CsGGPS9-2和CsGGPS9-3,在系统进化树上与其他基因分成两支。蛋白质序列分析表明,茶树GGPS家族成员都具有polyprenyl_synt结构域,不存在信号肽序列。亚细胞定位预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4定位在叶绿体上,CsGGPS3和CsGGPS9定位在线粒体上。通过Swiss Model进行三维建模,结合"three-floor"模型对茶树GGPS家族成员的功能进行预测,预测结果显示,CsGGPS1、CsGGPS2和CsGGPS4是GGPS;CsGGPS3是异源二聚体形式的牻牛儿基焦磷酸合酶的小亚基;CsGGPS9的催化主产物是碳链数大于30的异戊烯基焦磷酸。q RT-PCR分析表明,CsGGPS1整体表达丰度较低,仅在一芽二叶中表达量稍高;CsGGPS2在茶树各个组织中均有表达,在花中表达量最高,且花发育过程中表达量先上升后下降;CsGGPS3在叶和幼根中的表达量高于花,花发育过程中表达平稳;CsGGPS4在茶树各个组织中表达量数值相近,在花发育过程中表达量变化趋势与CsGGPS2相同;CsGGPS9的表达量在成熟叶中显著低于幼嫩叶片。     

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.     

两种干燥方式对香椿粉品质特性的影响

以香椿为原料，研究真空冷冻干燥和喷雾干燥对香椿粉的物理特性、基本成分、抗氧化活性、风味物质和微观结构的影响。结果表明：真空冷冻干燥香椿粉的综合品质较好。在物理特性方面，真空冷冻干燥香椿粉的得率较高、色泽浓绿，复水性、吸油性好；而喷雾干燥香椿粉的溶解度高、堆积密度大。在基本成分方面，真空冷冻干燥香椿粉的蛋白质、皂苷、生物碱含量均高于喷雾干燥；而VC、黄酮、多糖含量在两种干燥方式的香椿粉中无显著性差异。在抗氧化活性方面，真空冷冻干燥香椿粉的DPPH自由基清除力和还原力均高于喷雾干燥。在风味物质方面，从真空冷冻干燥和喷雾干燥的香椿粉中分别鉴定出49和41种挥发性成分，且真空冷冻干燥香椿粉含有较高的特征风味物质2-巯基-3, 4-二甲基-2,3-二氢噻吩。扫描电镜观察微观结构发现真空冷冻干燥香椿粉呈现蜂窝状松散结构，而喷雾干燥香椿粉多呈现出干瘪的圆球体状。综合考虑，真空冷冻干燥是一种适合制备高品质香椿粉的干燥方式。     

基于转录组测序的半滑舌鳎长链非编码RNA的初步鉴定与分析

以抗病家系与易感家系半滑舌鳎为材料,进行哈维弧菌感染实验,并对易感家系感染前(CsSU)、易感家系感染后(CsSC)、抗病家系感染前(CsRU)、抗病家系感染后(CsRC)4组进行转录组测序,根据RNA-seq数据挖掘半滑舌鳎长链非编码RNA信息;通过生物信息学分析,筛选出与抗哈维弧菌病相关的差异长链非编码RNA(long non-coding RNA, lncRNA)。结果显示,共识别出4 584个lncRNA座位,包含5 714个转录本;其基本特征与编码基因的比较分析,lncRNA的GC含量低于编码基因,单外显子基因数多于编码基因,转录本的平均长度长于编码基因,基因表达量低于编码基因。对4组样品进行两两比较(CsRU vs CsSU、CsRC vs CsSC、CsRC vs CsRU、CsSC vs CsSU)分别筛选出818、813、261、140个差异表达lncRNA,其中CsRU与CsSU之间、CsRC与CsSC之间lncRNA数目差异最多,通过聚类分析确定了各实验组的表达模式之间的联系,CsRU与CsSU之间的表达模式最为相近。通过共表达分析,预测出lncRNA和274个编码基因可能存在14 539种相互关系,并进行了功能注释,进而筛选出7个关键lncRNA。qRT-PCR结果显示,差异表达lncRNAs的表达模式和转录组数据得到的基本一致。研究结果为揭示lncRNA在半滑舌鳎抗哈维弧菌免疫调控反应中的作用提供重要的参考数据。     

油-棉连作棉田油菜秸秆覆盖对棉田杂草发生及土壤杂草种子库的动态影响

在安徽省沿江棉区开展了油菜秸秆覆盖对棉田杂草发生、棉花生长及土壤杂草种子库影响的研究。结果表明,随着油菜秸秆覆盖量的增加,对棉田杂草的抑制效果增强。与未覆盖秸秆且不除草处理相比较,7000Kg/hm²秸秆覆盖量处理棉花单株铃数和子棉产量显著提高。7000Kg/hm²秸秆覆盖量处理在覆盖后30d、60d和120d逐步减少0-20cm土层杂草种子库密度,与全程除草剂处理较一致；随着覆盖量减少,对0-20cm土层杂草种子库密度降低幅度减小。全程除草剂处理降低0-5cm土层杂草种子库多样性,而油菜秸秆覆盖则可能增加0-5cm土层的杂草种子库多样性。3500Kg/hm²覆盖量+秸秆覆盖30d后喷施除草剂处理的抑草效果和增产效果与全程除草剂处理一致。因此,在安徽省沿江棉区油-棉连作棉田推荐使用3500Kg/hm²油菜秸秆覆盖量+秸秆覆盖30d后喷施除草剂。