Turbidity Assay for Rapid and Efficient Identification of High Protein Digestibility Sorghum Lines

Recently, our laboratory reported a protein digestibility assay based on SDS‐PAGE that distinguishes mutant high protein digestibility from wild‐type sorghum lines. Using that assay, high protein digestibility sorghum lines were identified both qualitatively (visual observation) and quantitatively by measuring the SDS‐PAGE band intensity of the undigested α‐kafirin protein. Here, we report on a new turbidity assay that can be used for an even quicker quantitation of the undigested proteins with much higher throughput for screening purposes. Proteins remaining after 1 hr of pepsin digestion were extracted with a buffer of SDS, 2‐mercaptoethanol, and borate and an aliquot of the extract was precipitated using 72% trichloroacetic acid (TCA). Absorbance of the resulting turbid solution was then read at 562 nm. Lower readings corresponded to more digestible lines. The turbidity of the suspensions developed quickly and reached a plateau at ≈5 min for high protein digestibility lines and 10 min for wild‐type lines. The turbid solutions remained stable for at least 1 hr. Two distinct groups, wild‐type and high protein digestibility sorghum lines, were obtained when the assay was compared with a standard pepsin digestibility procedure and to our recently developed SDS‐PAGE assay. A comparison with the bicinchoninic acid (BCA) assay of protein quantitation indicated that the turbidity assay is more efficient in differentiating between wild‐type and high protein digestibility sorghum lines. We have further refined the turbidity assay for microtiter plate analysis making it possible for a single operator to analyze ≈200 sorghum lines per day, compared to 60 lines when using the SDS‐PAGE assay.     

不同发育时期转基因高梁不同株系Cry1Ab蛋白含量的比较

高粱的害虫种类众多，影响着产量与品质，培育抗虫品种是重要的育种目标，但目前高粱中缺乏有效的抗螟虫资源，难以通过常规育种培育出抗螟虫的品种。张明洲（2002）成功地将来自Bt密码子优化的抗虫基因crylAb转入高粱中，并经农业部批准进入中间试验阶段。为有效和合理地利用这一抗虫资源，有必要了解crylAb基因的表达情况，本实验分析了田间自然条件下crylAb基因在不同发育时期的表达水平，并对不同转基因高粱株系的CrylAb蛋白含量进行了比较。     

A Novel Modified Endosperm Texture in a Mutant High‐Protein Digestibility/High‐Lysine Grain Sorghum (Sorghum bicolor (L.) Moench)

Development of high‐protein digestibility (HPD)/high‐lysine (hl) sorghum mutant germplasm with good grain quality (i.e., hard endosperm texture) has been a major research objective at Purdue University. Progress toward achieving this objective, however, has been slow due to challenges posed by a combination of genetic and environmental factors. In this article, we report on the identification of a sorghum grain phenotype with a unique modified endosperm texture that has near‐normal hardness and possesses superior nutritional quality traits of high digestibility and enhanced lysine content. These modified endosperm lines were identified among F₆ families developed from crosses between hard endosperm, normal nutritional quality sorghum lines, and improved HPD/hl sorghum mutant P721Q‐derived lines. A novel vitreous endosperm formation originated in the central portion of the kernel endosperm with opaque portions appearing both centrally and peripherally surrounding the vitreous portion. Kernels exhibiting modification showed a range of vitreous content from a slight interior section to one that filled out to the kernel periphery. Microstructure of the vitreous endosperm fraction was dramatically different from that of vitreous normal kernels in sorghum and in other cereals, in that polygonal starch granules were densely packed but without the typically associated continuous protein matrix. We speculate that, due to the lack of protein matrix, such vitreous endosperm may have more available starch for animal nutrition, and possibly have improved wet‐milling and dry‐grind ethanol processing properties. The new modified endosperm selections produce a range that approaches the density of the vitreous parent, and have lysine content and protein digestibility comparable to the HPD/hl opaque mutant parent.     

Low α-Amylase Starch Digestibility of Cooked Sorghum Flours and the Effect of Protein

The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize. Four sorghum cultivars that represent different types of endosperm were used. Starch digestibilities of 4% cooked sorghum flour suspensions, measured as reducing sugars liberated following α-amylase digestion, were 15–25% lower than for cooked maize flour, but there were no differences among the cooked pure starches. After the flours were predigested with pepsin to remove some proteins, the starch digestibility of cooked sorghum flours increased 7–14%, while there was only 2% increase in normal maize; however, there was no effect of pepsin treatment on starch digestibility if the flours were first cooked and then digested. After cooking with reducing agent, 100 mM sodium metabisulfite, starch digestibility of sorghum flours increased significantly while no significant effect was observed for maize. Also, starch solubility of sorghum flours at 85 and 100°C was lower than in maize, and sodium metabisulfite increased solubility much more in sorghum than in maize. Differential scanning calorimetry results of the flour residue after α-amylase digestion did not show any peaks over a temperature range of 20–120°C, indicating that sorghum starches had all undergone gelatinization. These findings indicate that the protein in cooked sorghum flour pastes plays an important role in making a slowly digesting starch.     

Impacts of Kafirin Allelic Diversity,Starch Content,and Protein Digestibility on Ethanol Conversion Efficiency in Grain Sorghum

Seed protein and starch composition determine the efficiency of the fermentation process in the production of grain‐based ethanol. Sorghum, a highly water‐ and nutrient‐efficient plant, provides an alternative to fuel crops with greater irrigation and fertilizer requirements, such as maize. However, sorghum grain is generally less digestible because of extensive disulfide cross‐linking among sulfur‐rich storage proteins in the protein– starch matrix. Thus, the fine structure and composition of the seed endosperm directly impact grain end use, including fermentation performance. To test the hypothesis that kafirin (prolamin) seed storage proteins specifically influence the efficiency of ethanol production from sorghum, 10 diverse genetic lines with allelic variation in the β‐, γ‐, and (δ‐kafirins, including three β‐kafirin null mutants, were tested for ethanol yield and fermentation efficiency. Our selected lines showed wide variation in grain biochemical features, including total protein (9.96–16.47%), starch (65.52–74.29%), and free amino nitrogen (FAN) (32.84–73.51 mg/L). Total ethanol yield (ranging from 384 to 426 L/metric ton), was positively correlated to starch content (R² = 0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (R² = 0.52). Increases in FAN content enhanced fermentation efficiency (R² = 0.65). The highest ethanol producer was elite staygreen breeding line B923296, and the line with the highest fermentation efficiency at the 72 h time point was inbred BT×623. A large‐seeded genotype, KS115, carrying a novel γ‐kafirin allele, was rich in FAN and exhibited excellent short‐term fermentation efficiency at 85.68% at the 20 h time point. However, the overall ethanol yield from this line was comparatively low at 384 L/metric ton, because of insufficient starch, low digestibility, and high crude protein. Multivariate analysis indicated an association between the β‐kafirin allele and variation in grain digestibility (P = 0.042) and FAN (P = 0.036), with subsequent effects on ethanol yield. Reversed‐phase HPLC profiling of the alcohol‐soluble kafirin protein fraction revealed diversity in protein content and composition across the lines, with similarities in peak distribution profiles among β‐kafirin null mutants compared with normal lines.     

不同发育时期转基因高粱不同株系Cry1Ab蛋白含量的比较

高粱的害虫种类众多,影响着产量与品质,培育抗虫品种是重要的育种目标,但目前高粱中缺乏有效的抗螟虫资源,难以通过常规育种培育出抗螟虫的品种。张明洲(2002)成功地将来自Bt密码子优化的抗虫基因cry1Ab转入高粱中,并经农业部批准进入中间试验阶段。为有效和合理地利用这一抗虫     

转基因产品中PAT蛋白的酶联免疫检测

建立并优化了转基因油菜(Brassica campestris )中的PAT蛋白酶联免疫检测技术。首先通过Western杂交和酶活性分析鉴定了PAT蛋白的纯度和活性。然后以其为抗原免疫新西兰大白兔制备了PAT蛋白的多克隆抗体，并采用硫铵沉淀法和protein A-Sepharose 4B对其进行了纯化。所得抗体对PAT蛋白的检测限为2×10-5 mg/mL，且与植物源的几种结构功能相关蛋白均无交叉反应。然后对植物蛋白进行初步提取，应用酶联免疫检测技术对转基因油菜(MS1/RF1 and MS8/RF3)中的PAT蛋白进行了检测，结果表明ELISA能高效地鉴别转基因和非转基因油菜.     

快速获得葫芦科核糖体失活蛋白新基因

根据葫芦科核糖体失活蛋白(ribosome-inactivating protein，RIP)上、下游两段高度保守的氨基酸序列设计简并引物对LYI／LY2，对基因组DNA进行PCR扩增，快速获得了3种RIP新基因，分别是冬瓜(Benincasa hispida)来源的benincasin(AF453777)、南瓜(Cucurbita moschata)来源的moschatin Ⅰ(AF462349)和moschatin Ⅱ(AF504011)。它们的编码区与葫芦科6个代表RIP对应区段的同源性分别为：trichosanthin(61％、69％和55％)、α-momorcharin(63％、72％和69％)、α-Iuffin(72％、83％和72％)、RIP from Cucumis figarei(80％、69％和79％)、sechiumin(43％、43％和44％)以及bryodin Ⅰ(62％、65％和58％)。葫芦科RIP的LYI／LY2区段存在43个完全一样的氨基酸残基，其中8个残基在其它科属来源的8个Ⅰ型和3个Ⅱ型RIP中也完全保守，包括构成活性中心的4个关键残基。     

Assessing Fermentation Quality of Grain Sorghum for Fuel Ethanol Production Using Rapid Visco‐Analyzer

The Rapid Visco‐Analyzer (RVA) was used to characterize the pasting properties of 68 sorghum grains with a standard 23‐min temperature profile. The results showed a strong linear relationship between ethanol yield and final viscosity as well as setback. Ethanol yield increased as final viscosity decreased. A modified RVA procedure (10 min) with an application of α‐amylase was developed to simulate the liquefaction step in dry‐grind ethanol production. There was a remarkable difference in mashing properties among the sorghum samples with the normal dosage of α‐amylase. The sorghum samples which were difficult to liquefy in the mashing step had much higher peak viscosities than the samples that were easily liquefied. The results also showed that the relationship between conversion efficiency and mashing property was significant. Tannins cause high mash viscosities. There was a strong linear relationship between tannin content and final viscosity as well as peak viscosity. The modified RVA procedure is applicable not only for characterization of mashing properties but also for optimization of α‐amylase doses for starch liquefaction.     

AS-PCR技术检测鸡EX-FABP基因型方法的建立

摘要： 鸡(Gallus gallus )胞外脂肪酸结合蛋白(extracelluar fatty acid binding protein, EX-FABP)基因型与鸡腹脂量的积累密切相关。尝试了用等位基因特异性PCR(allele-Specific PCR, AS-PCR) 技术检测EX-FABP基因型的方法，确定了检测的参数和方案, 对AS-PCR检验单碱基突变的方法和策略进行了讨论,并在此基础上对该技术进行了改进，建立了等位基因特异性片段长度差异PCR（allele-specific and length-different PCR, ASLD PCR）技术。     

抗苯巴比妥单克隆抗体杂交瘤细胞株的筛选及ciELISA试剂盒的研制

用BSA-pAPB免疫Balb/c小鼠，用细胞融合技术制备并用间接ELISA和阻断ELISA筛选抗苯巴比妥单克隆抗体(PB mAb)杂交瘤细胞株，体内诱生腹水法生产PB mAb，应用PB mAb研制PB残留竞争ELISA(ciELISA)快速检测试剂盒(PB-Kit)，并测定其性能。结果表明，筛选出3株杂交瘤细胞，最好的3F6-C4株的PB mAb间接ELISA效价为1∶6.4×105，亲和常数(Ka)为1.96×1010 L/moL，半数抑制浓度(IC50)为5.7 μg/L，与巴比妥的交叉反应率(CR%)12.4%，与其它化合物无CR；PB-Kit的线性检测范围1.0～81 μg/L，灵敏度0.75 μg/L，检测限1 μg/L；饲料样和猪尿样的平均添加回收率85.8%和91.3%，平均批内和批间变异系数均<15%。PB-Kit具有快速、敏感、特异、简便等特点，适合PB残留快速检测的推广应用。     

玉米多胞质系及其遗传分析Ⅰ.多胞质系的非酶形式蛋白质生化标记

已培育出一组多胞质系,即含Mo17细胞核的同核异质系.在不同发育阶段,对11种多胞质系的不同组织进行了蛋白质电泳分析,结果筛选到生化标记,从而可以在生化水平上将不同的多胞质系予以区分.     

Rapid Size‐Exclusion Chromatography Analysis of Molecular Size Distribution for Wheat Endosperm Protein

A quick method for the separation of the main size classes of wheat endosperm proteins using size‐exclusion HPLC is presented. Separations achieved in a 10‐min run showed high correlation with the reference method of our laboratory (35‐min) using the same type of column. The clear separation obtained allows a quick analytical determination of important parameters such as the proportion of the main classes of wheat endosperm protein, the glutenin‐to‐gliadin ratio, and the percentage of unextractable (SDS‐soluble with sonication) polymeric protein, all of which have been closely correlated with breadmaking quality parameters. The improved method offers the advantages of better utilization of valuable resources such as HPLC equipment, quicker analysis of large sample sets, and collection of eluted fractions.     

一种高效制备慢病毒表达载体的方法

慢病毒表达载体具有感染宿主广和基因组整合效率高的优点,因此在转基因动物的制备中得到广泛应用.本研究构建了含有红色荧光和绿色荧光的慢病毒表达载体,用磷酸钙转染法将三质粒慢病毒载体转染293T细胞,转染效率达72%,降低了慢病毒的生产成本.在进行慢病毒的浓缩与纯化时,通过超速度离心和超滤相结合的方法,在150 mL的慢病毒原液中经纯化后得到80 μL效价为2.38×109 TU/mL的慢病毒颗粒.获得的高效价双荧光慢病毒表达载体可为后续双基因转基因动物的制备提供可靠的制作途径与检测基础.     

Energy-Dispersive X-ray Fluorescence Spectrometry for Cost-Effective and Rapid Screening of Pearl Millet Germplasm and Breeding Lines for Grain Iron and Zinc Density

Comparison of energy-dispersive X-ray fluorescence (XRF) and inductively coupled plasma-optical emission spectroscopy (ICP) for iron (Fe) and zinc (Zn) densities in pearl millet grain samples from 11 trials showed significant differences between these two methods for both micronutrients. XRF values were more often higher than the ICP values for both micronutrients, but the differences were significant in only 15–38% genotypes for Fe and in 7–25% genotypes for Zn across the trials. In 82% genotypes the differences between these two methods were ≤6 mg kg^?1 for Fe; and in 88% genotypes, the differences were ≤4 mg kg^?1for Zn. There were highly significant and high positive correlations between ICP and XRF for both micronutrients. Selection of genotypes above the XRF trial mean for Fe/Zn included at least 30% top-ranking genotypes based on ICP. Therefore, XRF can be used for cost-effective and rapid screening of a large number of grain samples in pearl millet biofortification programs.     

A New Microbial Contact Assay for Marine Sediments (7 pp)

-  Dedicated to Prof. Dr. Ulrich Förstner on his 65th birthdayBackground, Aims and Scope   The number of microbiological contact tests for marine sediments is low, although microorganisms enable a rapid screening and monitoring of sediment quality and a high resolution of hazard assessment. As no single biotest can provide reliable answers concerning the potential hazard of environmental samples, a combination of bioassays needs to be applied to serve this purpose. In order to cover as many potential effects as possible, test organisms should have different sensitivities; assays should cover different exposure pathways, and measure the effect on various physiological functions. Methods   3 different Vibrio species (V. proteolyticus, V. natriegens, V. gazogenes) were tested for their suitability as test organisms in a contact assay on the basis of their activity, sensitivity and their spectrum of salinity tolerance. As a test endpoint, dehydrogenase activity over an incubation time of 2 hours (~ 3 generations) was chosen, quantified by resazurine reduction. The test was miniaturized to 96 well plates, including a dilution series, and quality criteria were established. The assay was then tested on natural sediments from a contaminated site in the Lübeck Bight.Results and Discussion   Vibrio proteolyticus proved to be the best suited test organism out of those tested for this bioassay. The miniaturized test system revealed a coefficient of variation of positive controls in 16 tests of 17.8 %. Its application to contaminated sediments from the Lübeck Bight showed a good differentiation of samples from different depths and zones, which reflected the general contamination pattern and capping activities in that area. Conclusion   The miniaturized test system that has been developed for V. proteolyticus is suited to assess toxic effects of brackish and marine sediments. Due to an increased number of replicates and an extensive sediment dilution series, the degree of certainty of hazard assessment is elevated.Recommendations and Outlook   The integration of this sediment contact assay as a complementary test in a microbiological test battery is recommended.     

病毒加速传播机制的新发现

病毒的传播被认为是易感细胞的反复感染过程,通过复制、释放不断传播,如果真是这样的话,传播速度会被复制动力学所限制.但是研究人员发现牛痘病毒传播1个细胞仅需75 min,比其复制周期快4倍.为了解释这一现象,英国帝国理工学院研究人员进行了相关研究.研究表明,有些病毒会刺激受感染细胞,使其把病毒当作乒乓球弹来弹去,这样可以大大加快病毒感染其他细胞的速度.     

白血病中C-myb蛋白的作用

据《科学日报》（ScienceDaily）2010年1月19日报道,美国费城宾夕法尼亚大学医学院的Alan Gewirtz和他的同事现在已经鉴定了一个对诱导MLL关联的白血病的形成起重要作用分子途径。     

魔芋DNA快速微量提取及其ISSR-PCR扩增

魔芋属天南星科魔芋属草本植物,其组织富含多糖和多酚等次生物质,这类物质的存在不仅使DNA提取变得困难,还会影响下游的分子生物学操作。采用改进的提取方法,主要包括在2mL Eppendorf离心管中液氮研磨,CTAB-SDS裂解细胞,β-巯基乙醇浓度从2％提高到5％和添加PVP抑制多酚氧化,缓冲液去多糖等步骤,成功的提取了魔芋鲜叶的高质量DNA,其A260／A280位于1．80-2．00之间,产率超过470μg／g,ISSR0PCR扩增效果好。该方法具有快速、低廉、微量、稳定等特点,为深入研究魔芋资源遗传多样性、标记辅助育种和种芋纯度的鉴定奠定了基础。     

猪伪狂犬病gE-ELISA鉴别诊断方法的建立及初步应用

摘要: 为了区分伪狂犬病野毒感染猪与糖蛋白E(gE　)基因缺失疫苗免疫猪，利用大肠杆菌(Eschirchia coli　)BL21(DE3)表达伪狂犬病病毒gE糖蛋白，经纯化、变性、复性等处理后将gE蛋白作为抗原建立了伪狂犬病的gE-ELISA鉴别诊断方法。以该方法检测115份已知背景猪血清，结果表明，该方法诊断特异性为94.5%，诊断敏感性为96.7%。以5块不同批次的包被抗原的酶标板检测5份血清，结果显示阳性血清的变异系数均小于10%，表明该方法重复性好。对比国外阻断gE-ELISA同时检测临床356份猪血清，两者阳性符合率为87.44%（195/223），总符合率为92.13%（328/356）。以上结果表明该gE-ELISA特异、敏感且重复性好，可用于猪伪狂犬病的鉴别诊断。