四种海藻热休克蛋白70（HSP70）基因的克隆与表达分析

热休克蛋白70是热休克蛋白家族中重要的成员，参与新生蛋白的折叠、转运、重折叠变性蛋白、协助降解变性蛋白和抗逆环境胁迫等功能。海带和裙带菜是浅海潮下带典型的褐藻，孔石莼和浒苔是潮间带典型的绿藻，四种大型海藻均有重要的经济价值和生态价值。随着潮汐变化固着藻类生境理化因素变化剧烈，藻类面临着严重的环境胁迫，因此研究藻类抗逆机理有着重要的意义。 本研究采用同源克隆法配合RACE-PCR，克隆了海带、孔石莼、裙带菜和浒苔HSP70基因的全序列（分别命名为LJHSP70、UPHSP70、QDHSP70和EPHSP70）。利用生物信息学方法分析了四种藻类HSP70结构特征、同源性关系和进化地位。获得的海带HSP70基因全序列长为2918 bp，5’非翻译区为248 bp，3’非翻译区为696 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.97。获得的裙带菜HSP70基因全序列长为3243 bp，5’非翻译区为248 bp，3’非翻译区为1021 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.96。获得的孔石莼HSP70基因全序列长为2283 bp，5’非翻译区为65 bp，3’非翻译区为247 bp，开放阅读框为1971 bp，编码656个氨基酸，预测的分子量为71.13 kDa，等电点为5.04。获得的浒苔HSP70基因全序列长为2265 bp，5’非翻译区为65 bp，3’非翻译区为217 bp，开放阅读框为1983 bp，编码660个氨基酸，预测的分子量为71.39 kDa，等电点为5.03。四种海藻HSP70氨基酸序列均含有四肽重复序列GGMP，具有三个典型的HSP70签名基序。细胞质定位的HSP70 C-末端特征基序为EEID或EEVD，并且N-端氨基酸序列保守性高于C-端。海带和裙带菜HSP70蛋白同源性为98%，孔石莼和浒苔HSP70蛋白同源性为96%，四种海藻HSP70蛋白序列与陆地植物和其他藻类HSP70蛋白序列同源性为70-80%。 利用荧光定量RT-PCR技术对不同胁迫条件处理的海带和孔石莼HSP70 mRNA的表达水平进行定量分析。不同热激温度（5-40 ℃）处理组中，30 ℃处理组的海带HSP70 mRNA表达量最高是10 ℃处理组的海带HSP70 mRNA表达量的3倍，而35 ℃或40 ℃处理组的海带HSP70表达量却低于25 ℃或30 ℃处理组的海带HSP70 mRNA表达量。25 ℃不同热激时间（0-12 h）处理组中，海带HSP70 mRNA表达量呈先上升后下降趋势。热激1 h后海带HSP70 mRNA表达量迅速上升，热激7 h后mRNA表达量达到最大，是对照组表达量的4倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的海带HSP70 mRNA表达量是30‰盐度处理组海带HSP70 mRNA表达量的3倍。35‰、40‰和45‰盐度处理组之间HSP70 mRNA表达量较低且无显著差异。 不同热激温度（5-40℃）处理组中，20 ℃或25 ℃处理的孔石莼HSP70 mRNA表达量较低，而5 ℃、35 ℃、或40 ℃处理组的孔石莼HSP70 mRNA的表达量是25 ℃处理组孔石莼HSP70 mRNA表达量的2倍以上。30 ℃不同热激时间（0-12 h）处理组中，孔石莼HSP70 mRNA表达量也呈先上升后下降趋势。热激5 h后孔石莼HSP70 mRNA表达量达到最大，是对照组的3.5倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的孔石莼HSP70 mRNA表达量是30‰盐度处理组孔石莼HSP70表达量的3倍。30‰、40‰和45‰盐度处理组孔石莼HSP70 mRNA表达量较低，且无显著差异。不同紫外线照射时间（0-4.0 h）和不同干燥时间（0-4.0 h）处理组中，孔石莼HSP70 mRNA表达量都在3 h后达到最高值，之后表达量维持在较高水平。 为进一步研究藻类HSP70的生物学功能，将海带HSP70基因的开放阅读框区域克隆到表达载体pEASY-E2中，并转化到大肠杆菌BL21(DE3)pLysS。将阳性重组子培养于含有AMP（100 U/mL）的LB培养基，IPTG诱导表达，SDS-PAGE电泳鉴定。经5 h诱导，其表达量达到平台期，继续培养HSP70表达量并不显著增高。5 mM IPTG诱导海带HSP70蛋白表达量高于1 mM IPTG诱导蛋白表达量。

Comparative researches on effects of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate upon Lateolabrax japonicus biomarker system

Fish Lateolabrax japonicus were exposed to anion surfactant sodium dodecylbenzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS) at 1 mg/l, respectively, for 6, 12 and 18 d, with one control group. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione S-transferase (GST) were determined; brain acetylcholinesterase (AChE) and liver inducible nitric oxide synthase (NOS) activities were also measured. The results of the study indicated that these parameters made different, sometimes, adverse responses to SDBS and SDS exposure, such as the activity of NOS can be inhibited by SDBS and induced by SDS, the different physico-chemical characteristics of SDBS and SDS should be responsible for their effects on enzyme activities. (c) 2005 Elsevier B.V. All rights reserved.

Effects of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate on the Mytilus galloprovincialis biomarker system

The effects of in vivo exposure of Mytilus galloprovincialis to two anionic surfactants (SDBS and SDS) on the molecular biomarker system were studied. After continuous exposure for 72 days, activities/levels of GST, GPx and GSH were significantly higher than in corresponding control groups following exposure to 3.000 mg/L SDS and SDBS. Activities of SOD and CAT were significantly inhibited by experimental SDBS (except CAT in 0.100 mg/L group), but not by SDS. Statistical analysis of enzyme activities/levels suggested that there were significant positive relationships between GST and GPx, and negative relationships were found between GSH and CAT, GSH and SOD. Amplified fragment length polymorphism (AFLP) results showed that a greater genotoxic effect was observed for SDBS than for SDS. Based on the above results, the biomarker system of mussels can be affected by the two anionic surfactants (>= 3.000 mg/L); it was more easily affected by SDBS than by SDS. Crown Copyright (C) 2009 Published by Elsevier Inc. All rights reserved.

Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU50 against chicken erythrocytes was 0.91 A mu g/mL.

小麦高分子量谷蛋白亚基表达量与面团流变学特性的关系

应用SDS-PAGE技术依据HMW-GS组成型从来自全国10个省（区）、市的小麦品种（系）中筛选出20个,分为5组。准确提取各品种的高分子量谷蛋白,并将SDS-PAGE谱带用凝胶成像系统（Bio-imagingSystem）和Labworks5.0进行亚基定量分析,并进行了面团流变学特性测定,所得数据用SPSS14.0进行统计分析。结果表明,HMW-GS总表达量在各品种间的差异达到极显著水平,所有品种中各亚基的平均表达量10〉7〉5〉1〉8〉12〉2〉9;HMW-GS总表达量及HMW-GS占籽粒蛋白质含量的比例与面团形成时间、稳定时间和粉质评价值之间呈显著正相关;分组比较单个亚基对品质性状的影响表明,亚基1优于null,5＋10优于2＋12,7＋8和7＋9相当。研究发现不具有优质亚基组成、但HMW-GS表达量高的品种品质也较好,因此在小麦品质育种中,在注重HMW-GS组成筛选的同时,要注重保留HMW-GS表达量高的材料。

青海糯性春小麦综合标记辅助育种研究

选用全糯小麦品种‘糯麦1号’与青海主要栽培品种‘阿勃’杂交，综合利用改良碘染色法、SDS-PAGE法和Waxy基因的分子标记等，对杂交后代进行了鉴定筛选。最终从F2代鉴定出了5粒全糯种子，从F_代鉴定出8株Wx-B1亚基缺失的植株。对全糯株系F_3代的农艺性状进行评价，5个全糯株系的综合农艺性状都优于‘糯麦1号’，与‘阿勃’较接近。测定全糯株系和Wx-B1亚基缺失植株F_4代种子的直链淀粉含量，5个全糯株系F_4代种子的直链淀粉含量接近于0，8个Wx-B1亚基缺失植株F_4代种子的直链淀粉含量在总 体上比‘阿勃’的直链淀粉含量低。研究表明，采用综合标记辅助选择可快速而准确地获得适合在青海栽培的全糯和部分糯性小麦。

青海春小麦高分子量谷蛋白亚基分子标记辅助回交选择

为了改良青海省现有小麦主栽品种的品质性状，以青海省春小麦主栽品种青春533和高原602为轮回亲本，以具有1Dx5＋1Dy10基因且综合性状良好的宁春4号为非轮回亲本，利用与1Dx5基因共分离的PCR分子标记对BC_1F_1、BC_2F_1和BC_3F_1植株进行目标基因检测；将筛选到的具有1Dx5基因的BC_3F_1植株进行自交，获得了BC_3F_2种子，利用SDS-PAGE电泳对BC_3F_2种子进行HMW-GS分析，分别从青春533×宁春4号和高原602×宁春4号的BC_3F_2中筛选出5粒和2粒具有纯合5＋10亚基的种子，且中选植株的农艺性状基本接近轮回亲本。

野生大麦与青稞高分子量谷蛋白亚基遗传变异研究

通过SDS-PAGE方法对33份青稞Ⅰ组和5份野生大麦H组染色体编码的高分子量谷蛋白亚基（HMW—GS）的多态性进行了研究。结果表明Ⅰ组和H组染色体编码的HMW-GS亚基之间存在带型差异，Ⅰ组高分子量谷蛋白亚基存在两种带型，一种接近7亚基上部，一种接近7亚基下部，H组内部只有一种带型，靠近10亚基。因此，要改进青稞的面筋品质现状，应在青稞中引入新的优质HMW—GS亚基的变异类型。

弱筋小麦品种高分子量谷蛋白亚基组成分析

为了有效地利用从国内其他地区引进的低蛋白弱筋种质材料,采用SDS-PAGE技术对63份材料进行了高分子量谷蛋白亚基组成的分析.结果表明,参试材料中共有14种HMW-GS类型,Glu-A1位点上有Null、1和2~*三种类型,以Null为主(52.4%);Glu-B1位点上有13+16、17+18、7、7+8、7+8+9和7+9六种类型,以7+8为主(55.6%);Glu-D1位点上有10、12、2+12、5+10、5+12五种类型,以2+12为主(61.9%),而5+10为27%.亚基组合类型共有22种,以"1,7+8,2+12"为主(22.2%).品质评分频率最高的是8分,为38.1%,其次为6分,为14.3%,5分的为9.5%,但品质评分为10分的也有5个材料,频率为7.9%.优质亚基含量等同或高于其他同类研究,品质评分也相对较高,这说明我国弱筋小麦的选择,需要加强对HMW-GS组成的分析.

青海省春小麦收割后田间堆放期间产量与品质的变化

为了解青海省春小麦收割后田间堆放期间产量和品质的变化，选择近年育成的春小麦品种“高原205”（白粒，易穗发芽）、“高原115”（紫黑色小麦）、“高原448”（红粒）和“高原314”（白粒）以及曾为青海省主栽品种的“青春533”（红粒）为材料，收割后以农民常用的田间堆放方式进行堆放，每隔9d对其进行采样，测定产量、SDS沉淀值和降落数值的变化。结果表明，随着堆放时间的延长，收获产量逐步下降，平均产量从第1次采样（Od）时的504g／m^2减少到63d后的384g／m^2，收获产量平均值与取样时间直线回归F值为20．91，大于F0.01（6．90）；SDS沉淀值变化较小，F值为0．90，小于F0．05（2．13）；降落数值在取样时间点上差异极显著．F值等于46，72，大于F0.01（2．87）。高原205的降落数值一直呈现下降趋势，而高原448、高原115、高原314和青春533的变化是在O～18d间上升，然后下降。总体而言，田间堆放对收获产量和品质不利。

红景天中红景天甙和酪醇的毛细管电泳法分析

利用毛细管电泳法分离测定两种红景天中红景天甙和酪醇的含量,所用毛细管规格为48.5 cm×50 μm, 二极管阵列紫外检测器 (DAD)检测波长221 nm, 最佳分离条件:电压21 kV, 分离温度25 ℃,背景电解质为含有30 mmol/L 十二烷基硫酸钠(SDS), 2.5+97.5(V/V)乙腈的14 mmol/L硼酸溶液,pH 10.7.红景天甙与酪醇分别在60.0～7.5 μg/mL 和 27.5～3.5 μg/mL质量浓度范围内与电泳峰面积呈现良好线性关系,检测下限分别为3.0和1.5 μg/mL .对标准品进行6次测定, 迁移时间的RSD为0.25%和0.39%, 峰面积的RSD为5.26%和3.52%.

青海高原不同海拔地带生长的珠芽蓼光合特性的比较

对生长于青海高原不同海拔的珠芽蓼( Poligonum viviparum) 叶片的光合特性进行比较研究发现：随海拔升高叶绿素含量有降低趋势,叶绿素a/ b 比值及类胡萝卜素含量有增大趋势。不同海拔的珠芽蓼叶片和叶绿体的Fv/ Fo 、Fv/ Fm 和Rf d 值随海拔升高而增大, 表明随海拔升高, 其潜在光合活力增加。低温荧光测定显示,随海拔升高叶绿体中PSI 和PSI I 的相对荧光产量比值(PSI/PSI I) 减小。叶绿体蛋白质SDS - PA GE 结果表明,在28 - 32KD 附近电泳谱带的变化显著

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd tauri and Pd strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminat domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species. (C) 2007 Elsevier Ltd. All rights reserved.

Separation and determination of flavone and xanthone glycosides in Tibetan folk medicinal species Swertia mussotii and S-franchetiana by capillary electrophoresis

The contents of five pharmacologically active flavone and xanthone glycosides, namely, swertianolin, swertisin, isoorientin, mangiferin, and 7-O-[alpha-L-rhamnopyranosyl-(1 -> 2)-beta-D-xylopyranosyl]-1,8-dihydroxy-3-methoxyxanthone, extracted from Tibetan folk medicinal species Swertia mussotii and S. franchetiana were determined by capillary electrophoresis with diode-array detection. The separation of five components has been optimized with a capillary column with a total length of 48.5 cm and effective length of 40 cm (50 mu m i.d). The influence of the running buffer, the sodium dodecyl sulfonate (SDS) concentration, organic modifier, etc. on the resolution was evaluated. The background electrolyte contained 30 mM borate buffer, 28 mM SDS, 1.0% (v/v) acetonitrile, and was adjusted to pH 9.0 with 0.1 M NaOH. A good baseline resolution was obtained for the separation of five components within 5 min with the working voltage of 24 kV and a column temperature of 25 degrees C. The established method was rapid and reproducible for the separation and determination of five flavone and xanthone glycosides from the extracts of S. mussotii and franchetiana plant samples.

CE determination of 2-(9-carbazole)ethyl chloroformate-labeled oligopeptides

A pre-column derivatization method for sensitive determination of oligopeptides, using the tagging reagent 2-(9-carbazole)ethyl chloroformate (CEOC-Cl) followed by capillary electrophoresis (CE) with diode-array detection, has been developed. Maximum yield close to 100% were observed when a three to fourfold molar excess of reagent was used at pH 9.0-10.0. Excess reagent was extracted with n-hexane-ethyl acetate 9:1-10:1 (v/v); this enabled direct analysis using CE with no significant disturbance from the major fluorescent reagent degradation by-products. The effects on the results of buffer pH and of SDS and organic modifier concentrations were examined. Good baseline resolution in the separation of five CEOC-peptides was achieved with a 48.5-cm total length (effective length 40 cm) 50-mu m inner diameter capillary column.