The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

A kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.     

水稻着丝粒特异组蛋白CENH3抗体的制备与应用

【目的】着丝粒是真核生物染色体的基本功能元件之一,其功能是在细胞有丝分裂和减数分裂时期精确地调控染色体配对和分离并维持染色体的稳定。着丝粒结构是由DNA和蛋白质形成的一种复合体。着丝粒特异组蛋白(centromere-specific histone H3,CENH3)是功能着丝粒是否具有活性的最基本特征。所以制备CENH3的相关抗体是进行着丝粒结构与功能研究的前提条件之一。【方法】通过设计短肽进行兔免疫实验,制备了水稻着丝粒特异组蛋白CENH3兔源抗体,利用ELISA和蛋白免疫荧光(immunofluorescence,IF)等检测方法对抗体有效性进行了鉴定。【结果】ELISA检测显示制备的CENH3抗体有效稀释度为1:40万,并且蛋白免疫荧光信号在水稻体细胞每条染色体的着丝粒区域均能检测到。同时,该抗体也可以应用于玉米等其他物种。通过染色质免疫沉淀(ChIP)技术获得与CENH3相结合的DNA分子,并进行PCR扩增和FISH定位分析,结果显示相应的Ch IP-DNA位于水稻功能着丝粒区域。【结论】本研究制备的水稻CENH3兔源抗体能满足着丝粒研究中相关实验的要求,可进一步应用于着丝粒的结构与功能研究。     

Continuous Spectrophotometric Assay and Properties of Ascorbic Acid Oxidising Factors in Wheat

A continuous spectrophotometric assay was developed to measure ascorbic acid oxidation in crude Na₂SO₄ extracts of flour. The rate of ascorbic acid oxidation in flour extracts measured using this method was similar to the rate in flour-water suspensions and 2–4 fold less than the rate in dough measured using an indophenol-xylene extraction method. Flour extracts appeared to contain two ascorbic acid oxidising factors; one with optimal activity at pH 6·3 and 30 °C and the other with optimal activity at pH 10 and 40 °C. The pH 6·3 factor had properties similar to those of ascorbate oxidase (EC 1·10·3·3) in its pH and temperature stability, strong inhibition by NaN₃, KCN and diethyldithiocarbamate, inactivation by proteases, and greater stereospecificity towards -ascorbic acid than -isoascorbic acid. The pH 6·3 factor was most concentrated in the pollard milling fraction of wheat and was lowest in flour. The pH 10 factor had several properties indicating non-enzymic oxidation of ascorbic acid; it was not inactivated by proteases, it was inhibited poorly or not at all by the above ascorbate oxidase inhibitors, and it had low specificity for stereoisomers of ascorbic acid.     

昆虫抗真菌肽基因的分泌型表达及活性鉴定

 【目的】黑腹果蝇(Drosophila melanogaster)抗真菌肽Drosomycin (Drs)及其同系物Drosomycin-like C (Drs-lC)和斑腹刺螠蝽(Podisus maculiverntris)抗真菌肽Thanatin对丝状真菌具有广谱高效的抑杀作用。实现抗真菌肽基因在酵母中的高效分泌型表达，对探讨利用转基因酵母的发酵液直接进行果蔬、食品和农产品防腐保鲜的生物技术研发有重要意义。【方法】将Drosomycin基因（Drs）及其同系物基因Drs-lC和Thanatin基因分别与酵母分泌型表达载体pPICZαA重组，构建成重组表达质粒pPICZαA-Drs、pPICZαA-Drs-lC和pPICZαA–Thanatin。利用电转化将重组质粒转化毕赤酵母GS115，经表型筛选和PCR鉴定获得的重组毕赤酵母转化子，在甲醇诱导下进行抗真菌肽的分泌表达。【结果】3种抗真菌肽基因的酵母表达产物对6个供试真菌中的5个有明显的抑制作用，Thanatin同时对供试细菌有抗菌活性。【结论】Drs、Drs-lC和Thanatin抗真菌肽基因成功地转化毕赤酵母，并实现其分泌型表达。     

抗犬瘟热病毒荧光标记单抗的制备和初步鉴定

1材料与方法 1．1材料 1．1．1病毒与细胞。犬瘟热病毒（CDV）、狂犬病毒（RV）、犬细小病毒（CPV）、犬副流感病毒（CPIV）、犬腺病毒（CAV）,非洲绿猴肾细胞（Vero）、犬肾细胞（MDCK）、猫肾细胞（F81）,犬瘟热病毒单克隆抗体CE3由军事医学科学院军事兽医研究所犬病研究中心提供。     

影响西瓜农杆菌介导的高效遗传转化效率的主要因子

以西瓜子叶为外植体材料,利用GUS染色瞬时表达方法研究农杆菌浓度,预培养、共培养时间,预培养条件以及添加乙酰丁酸酮(AS)对西瓜转化效率的影响,优化西瓜遗传转化参数,初步建立以MS BAP 2.0mg/L IAA 0.2 mg/L Hyg 10 mg/L Cef 300 mg/L为分化培养基,黑暗条件下预培养2 d,使用OD为0.3的菌液侵染10 min后,黑暗条件下共培养3 d,然后在25℃,光周期为16 h的条件下筛选抗性芽的遗传转化体系。     

伏马菌素B1单克隆抗体的制备及鉴定

 【目的】真菌毒素可导致严重的食品安全问题。本试验旨在制备可用于检测伏马菌素B1的特异性单克隆抗体。【方法】制备伏马菌素B1人工抗原,杂交瘤细胞法筛选杂交瘤细胞株制备腹水型单克隆抗体,并对其特性进行鉴定。【结果】筛选得到杂交瘤细胞株F3,分泌的单克隆抗体亚类为IgG1,轻链类型为κ链;10%变性聚丙烯酰胺凝胶电泳显示单抗蛋白重链分子质量约为50 kD,轻链分子质量约为25 kD;间接酶联免疫吸附法测定杂交瘤细胞F3细胞培养上清和腹水效价分别为1:3 200和1:51 200;亲和力常数为1.60×10-8 mol•L-1;IC50值可达3.589 ng•mL-1;对其它真菌毒素不存在交叉反应;免疫转印法鉴定抗体具有高特异性。【结论】本试验制备FB1单克隆抗体具有较好亲和力和高特异性,具有良好的应用价值。     

拟除虫菊酯类农药半抗原合成及酶联免疫分析

酶联免疫分析(ELISA)是农药残留检测的一种重要方法,具有方便快速、特异灵敏等优点.主要介绍了拟除虫菊酯类农药半抗原的合成方法,对拟除虫菊酯分子的环丙烷、氰基和芳香环进行修饰;对拟除虫菊酯水解产物进行修饰;增加一个连接臂作为半抗原.同时介绍了酶联免疫分析的研究进展,对拟除虫菊酯类农药半抗原的合成及酶联免疫分析进行了讨论和展望.酶联免疫分析在拟除虫菊酯类农药的残留检测中将起到越来越重要的作用.     

小鼠精原干细胞分离培养条件的优化

采用两步酶消化法获得5～7日龄昆明小鼠睾丸组织的单细胞悬液,比较差速贴壁法和Percoll不连续密度梯度法对精原干细胞(Spermatogonial stem cells,SSCs)的纯化效果,并利用MTT法检测不同生长因子及添加浓度对SSCs体外增殖的影响.结果表明5日龄小鼠和7日龄小鼠的睾丸细胞总数存在较大差异(P<0.05),但SSCs数目无显著差异(P>0.05);与Percoll不连续密度梯度法相比,差速贴壁法获得了较高的SSCs数量和纯化率,其纯化率平均85.06%;MTT法检测结果表明LIF因子的添加对SSCs的增殖具有促进作用,20 ng/mL GDNF+20 ng/mLbFGF+103 U/mL LIF的组合添加是昆明小鼠SSCs的体外培养增殖的最佳组合.     

菲、芘对蚕豆的氧化胁迫和DNA损伤

以菲和芘为多环芳烃(PAHs)的代表物,以超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量为指标,研究了菲、芘对蚕豆的氧化胁迫;利用彗星实验分析了菲、芘对蚕豆DNA的损伤效应;将蚕豆幼苗根经抗氧化剂维生素E预处理后,暴露于菲、芘污染,研究了DNA损伤与氧化胁迫间的关系。结果表明,供试条件下菲、芘污染导致蚕豆幼苗SOD、POD、CAT活性提高和MDA含量上升,且MDA含量与菲、芘浓度均显著正相关;蚕豆根尖细胞DNA损伤随菲、芘暴露浓度的升高而增大,0~50 mg·kg-1菲污染条件下彗星图像尾矩(TM)值从46.41μm(阴性对照)增加到122.04μm(50 mg·kg-1菲污染处理),增大了162.96%。50mg·kg-1芘暴露下TM值从阴性对照的44.30μm增至110.36μm,增大了149.21%。经抗氧化剂维生素E预处理,蚕豆的DNA损伤程度减小。综上可知,菲、芘对蚕豆产生氧化胁迫并造成根尖细胞DNA损伤,菲、芘诱导的DNA损伤与氧化胁迫有关。