大豆主要过敏原Gly m Bd 30K基因的克隆及其原核表达载体的构建

利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆GlY m Bd 30K的完整开放阅读框,与pET一28a载体连接,构建原核表达载体.结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体.该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸.该蛋白质的相对分子质量为42 758,等电点为5.08.序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600.克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础.     

大豆主要过敏原Gly m Bd30K蛋白单克隆抗体的制备与应用

利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合.采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化.采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体...     

大豆过敏原Gly m 4蛋白抗原表位特征预测

通过生物信息学相关软件分析大豆过敏原Gly m 4蛋白理化性质(Prot Param)、信号肽(Signal P 4.1 Server)、跨膜区(TMHMM Server V 2.0)、B细胞表位(DNAStar)、MHC-Ⅱ类分子的结合能力(Net MHCⅡ2.2)。结果发现:Gly m4蛋白稳定性较好,无信号肽与跨膜区,转角结构丰富;B细胞抗原表位预测表明,Gly m 4蛋白61~64、93~94、122~125、127~130、134~137区域是潜在B细胞抗原表位;MHC-Ⅱ类分子的结合力分析表明Gly m 4蛋白144~153区域及82~96区域是潜在T细胞抗原表位,同时发现HLA-DRB10701、HLA-DRB10101等位基因型人群对Gly m 4蛋白较敏感。Gly m 4蛋白抗原表位分析为大豆过敏原的低过敏原性改造提供参考依据。     

大豆GmERF6基因的原核表达及重组蛋白纯化

将大豆中已克隆的一个新的ERF转录因子基因(GmERF6)构建到原核表达载体pET28上,导入大肠杆菌Rosetta(DE3)中,对其进行IPTG诱导.结果表明:在IPTG浓度为0.3 mol·L-1,诱导时间为3h时,重组蛋白得到表达,分子量大约为30 kDa.SDS-PAGE电泳结果表明重组蛋白主要以包涵体形式存在...     

橡胶树肌动蛋白解聚因子原核表达及纯化

本研究根据HbADF序列设计引物扩增基因的编码区,并将其插入到原核表达载体pET28a上,成功构建重组质粒pET28a-HbADF。将重组质粒转化大肠杆菌BL21(DE3)宿主菌,经1 mmol/L IPTG诱导,获得相对分子量为20 ku的融合蛋白。表达蛋白以可溶和包涵体两种形式存在,通过亲和层析方法纯化可溶蛋白并获得HbADF融合蛋白,用抗HIS标签的单抗对纯化蛋白进行了Western blot鉴定。该结果为进一步研究HbADF蛋白特性及功能奠定基础。     

水稻黑条矮缩病病毒外壳蛋白基因S10的原核表达、多克隆抗体制备及应用

用RT-PCR方法从感染水稻黑条矮缩病毒(rice black-streaked dwarf virus,RBSDV)水稻中克隆该病毒的外壳蛋白基因S10,然后将此外壳蛋白基因再亚克隆到原核表达载体PET-32a中构建成重组原核表达载体pET32a-CP。将重组表达载体转化大肠杆菌BL21(DE3),经IPTG诱导,Ni+ NTA亲和柱纯化获得分子量约为76kD含硫氧还蛋白的融合蛋白。以纯化的重组蛋白为抗原免疫兔子制备RBSDV外壳蛋白的多克隆抗体,并用制备的多克隆抗体建立了可靠、灵敏、特异的检测RBSDV的免疫捕获RT-PCR及Dot-blot ELISA方法,为该水稻病毒病的诊断提供技术支持。     

甘蔗黄叶病毒P0基因的克隆与原核表达及抗血清制备

根据已发表ScYLV-P0基因系列设计特异性引物,应用RT-PCR技术从甘蔗病叶的mRNA扩增得到目的 DNA片段。以pET32a(+)为原核表达载体,构建重组表达质粒pET32a-P0。经过双酶切鉴定和DNA测序后,将重组表达质粒转入大肠杆菌BL21(DE3)pLySs,在30℃培养条件下IPTG诱导表达。通过SDS-PAGE电泳检测融合蛋白表达情况。表达结果显示,在该表达系统中,融合表达蛋白P0是以包涵体形式的蛋白存在;P0融合蛋白大小约45kDa,与P0开放阅读框的理论推算值29.991 kDa加上载体自身蛋白约18 kDa相符,用Ni2+-NTA琼脂糖亲和层析纯化融合蛋白,免疫家兔制备出抗血清,通过酶联法(ID-ELISA)测定本实验制备的ScYLY-P0抗血清工作浓度为1∶25000。     

大豆蚜内共生菌groEL克隆及表达水平分析

为了解大豆蚜(Aphis glycines Matsumura)传播的大豆花叶病毒(soybean mosaic virus,SMV)与其体内共生菌产生的病毒结合蛋白(Gro EL)之间的作用机制,运用RT-PCR技术克隆得到大豆蚜内共生菌groEL基因全序列,把该基因与pET21b载体重组后进行原核表达,经Ni-Agarose His亲和层析得到纯化的蛋白,并利用荧光定量PCR技术分析了饲毒不同时间、不同龄期大豆蚜内共生菌gro EL基因表达量的变化。序列分析表明:大豆蚜内共生菌groEL基因全长序列为1 647 bp,编码548个氨基酸,推测蛋白分子量和pI值分别为69 k Da和5.24,成功构建重组载体pET21bGro EL进行原核表达,Western-blot鉴定确定为目的蛋白,蛋白可溶性分析发现重组蛋白为包涵体。随饲食SMV时间的延长大豆蚜内共生菌groEL的表达量显著增加,且无翅成虫与有翅成虫内共生菌groEL的表达量显著高于其它4个若虫期。大豆蚜内共生菌groEL基因能在原核细胞中稳定、正确表达,并且大豆蚜在饲食SMV后会诱导其内共生菌gro EL的表达,从而增加SMV爆发流行的可能性。     

大豆7S蛋白β亚基基因RNAi表达载体构建

以大豆7S蛋白β亚基基因为靶基因(基因编号:AB030840),利用RT-PCR克隆得到大豆7S蛋白β亚基基因核心序列398 bp,构建了以抗除草剂基因BAR为筛选标记的安全植物RNAi表达载体BAR-7αp-β.分子生物学检测表明7S蛋白β亚基基因的表达载体构建成功.研究结果为应用RNA干扰技术降低大豆过敏原,提高大豆蛋白品质奠定了基础.     

香蕉束顶病毒海口分离物Rb蛋白的原核表达、纯化及多克隆抗血清制备

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的E.coli BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结果表明,表达产物大小为22.6ku,且主要以包涵体形式稳定表达。目的蛋白经Ni2+-NTA琼脂糖亲和层析纯化后免疫家兔并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的BBTV编码Rb蛋白发生特异性反应。间接ELISA法测定的抗血清效价大于1:250000。用抗血清对感病香蕉和健康香蕉进行检测,结果表明,抗血清对感病香蕉有很高特异性,最佳工作浓度为1:1500。     

大豆皂甙抗血浆脂质过氧化和红细胞保护作用的研究

用分光光度法测定大豆皂甙的总抗氧化能力(T-AOC)和抗活性氧能力(ROSSC),用八木变法测定血浆过氧化脂质(LOOn)含量,用硫代巴比妥酸(TBA)法测定血浆丙二醛(MDA)含量,用分光光度法测定红细胞溶血程度,以期研究大豆皂甙的体外抗脂质过氧化和红细胞保护作用.结果表明:大豆皂甙具有较强的体外总抗氧化能力和抗活性氧能力,可抑制血浆和红细胞膜脂质过氧化作用,减少红细胞溶血程度.说明大豆皂甙具有体外抗血浆脂质过氧化和红细胞保护作用.     

基于nSSR和cpDNA序列的城步峒茶群体遗传多样性和结构研究

采用简单重复序列标记(nSSR)与叶绿体DNA序列(cpDNA)分析技术,对城步峒茶群体进行了遗传多样性、遗传结构和遗传分化等研究。结果表明,15对nSSR引物在参试81份资源中共获得142个等位位点,平均每对引物9.47个,城步峒茶群体的观测杂合度(Ho)、期望杂合度(He)和Nei期望杂合度(Nei)分别为0.49、0.62和0.62,具有较高的遗传多样性。Structure分析将79份峒茶资源分成3个类群,但各类群的遗传背景较为复杂,没有明显的群体结构。F检验表明,城步峒茶群体的近交系数F_IS为正值(F_IS=0.177 5),群体间的遗传分化系数F_ST较小(F_ST=0.034 5),分化程度较低,基因流Nm较高(Nm=7.01)。3对cpDNA引物分别获得了473 bp(rbcL)、704 bp(matK)和320 bp(psbH-trnA)的片段序列,变异位点占总位点的比例分别为0.42%、0.71%和1.25%。将3个序列依次拼接,共产生了9个单倍型,单倍型数由多到少的居群依次为TXZ(6)、DZC(4)、DPS(4)、TYS(3)、HJZ(2),群体的单倍型多样性(Hd)和核苷酸多样性(π)分别为0.732和0.001 39。9个单倍型中,单倍型H1和H5处于进化网络图的中心节点上,并且包含资源数量最多,属于比较原始的单倍型。同时,nSSR和cpDNA的AMOVA分析结果基本一致,居群内的变异百分比分别达到96.69%和80.54%,城步峒茶的遗传变异主要存在于居群内。     

Effects of crop density and tillage system on grain yield and N uptake from soil and atmosphere of sole and intercropped pea and oat

Pea (Pisum sativum L.) and oat (Avena sativa L.) were grown as sole and mixed crops in various densities under two different tillage systems on a loess soil near Göttingen/Germany in a 2-year field experiment (2002/2003). In the conventional tillage system a mouldboard plough (CT) was used and in the minimum tillage system a rotary harrow (MT) was employed. The effect of crop density and tillage system on the grain dry matter and grain N yields, N₂ fixation and soil N uptake were determined to address the following questions: (i) which mixture compositions exhibit the highest grain yields compared to the sole crops, (ii) which mixture compositions also fix a high level of N₂ and leave low levels of residual inorganic soil N after harvest, and (iii) whether the intercrop advantage is influenced by the tillage system. For (i) the result in 2002 showed that the highest grain yields of both sole cropped pea and oat and intercropped pea and oat were achieved at the highest densities. In 2003, when the inorganic soil N content was higher and weather conditions were warmer and drier, grain yields were significantly higher than in 2002, but sole as well as intercropped pea and oat gave their highest grain yields at lower densities. For both years and tillage systems, the highest intercrop advantages were achieved in mixtures with densities above the optimal sole crop densities. The result for (ii) was that a distinctly higher proportion of nitrogen was derived from the atmosphere (Ndfa) by intercropped pea than by sole cropped pea. However, the uptake of soil N by intercropped pea and oat was not reduced in comparison with that of sole cropped oat as the decrease in the uptake of N from the soil by oat at lower oat densities in the mixture was compensated for by the soil N uptake of pea. Additionally, the N_min-N content of the soil following the mixtures and sole cropped oat did not differ, especially in the deeper soil layers because oat in mixture was forced to take up more soil N from deeper layers. Therefore, the risk of soil N losses through leaching after mixtures was lower compared to sole cropped pea. The tillage system (iii) had no significant influence on grain yield and soil N uptake, but N₂ fixation and the competitive ability of intercropped pea were higher under CT than with MT. An additional result was that intercropping led to a significantly increased grain N content of both pea and oat compared to the sole crops. The increase in grain N content from sole to intercrop was from 3.30 to 3.42% for pea and from 1.73 to 1.96% for oat as a mean for both years and tillage systems. The present study confirms that growing pea and oat as intercrops highlights potential economic and environmental benefits which still need to be understood in more detail in order to exploit intercropping to a greater extent.     

乌龙茶品种（系）遗传多样性与亲缘关系的SSR分析

利用40对具有多态性的SSR引物对我国46份乌龙茶品种（系）的遗传多样性和亲缘关系进行了研究。结果表明,40对引物在供试材料中共检测到179个等位基因,329个基因型,平均检测4.48个等位基因,8.23个基因型,平均多态性信息量（PIC）为0.52。46份供试品种（系）的基因多样性指数（H）为0.57,观测杂合度（Ho）为0.40,遗传距离为0.59。按照地理来源分组分析表明,地区间乌龙茶品种（系）的遗传多样性以广东最高,其次为福建,最低的为台湾。亲缘关系分析表明,大部分品种（系）都按照其地理来源和遗传背景进行了聚类。根据育种方式分组的分析表明,杂交选育的品种（系）遗传多样性水平较其他方式选育的品种（系）低。     

亚麻CAD基因克隆及反义表达载体的构建

本试验以亚麻(Linum usitatissimumL.)品种Argos为材料,分离了高纯度的RNA。用简并引物以RT-PCR的方法,克隆了亚麻CAD基因cDNA部分序列,长度为477bp,构建了pGEM-T-CAD质粒。用限制性内切酶EcoRI酶切pGEM-T-CAD质粒和载体pGEM-7Zf(-),进行连接,构建成中间表达载体pGEM-7Zf(-)-CAD质粒。用限制性内切酶XbaI和BamHI双酶切中间表达载体pGEM-7Zf(-)-CAD质粒和pBI121载体,进行连接,构建了亚麻CAD基因反义植物表达载体pBI121-antiNTCAD。     

Occurrence of filamentous fungi and mycotoxins in wrapped forages in Sweden and Norway and their relation to chemical composition and management

A study was conducted on 124 farms in Sweden and Norway where fungal (all farms) and mycotoxin (100 of the 124 farms) presence was examined in wrapped forage bales. Samples were also analysed for chemical composition, and data on the bale production and storage system on each farm were collected. Fungi, analysed by three common sampling methods, were found in bales from 89% of the visited farms (110 farms). The most frequently isolated fungal species from the bale surface was Penicillium roqueforti. Mycotoxins were present in 39% of the 100 samples analysed. The most common mycotoxins present were enniatin B (14 farms) followed by deoxynivalenol (12 farms). The risk of finding fungi in baled forage samples was higher with odds ratio (OR) of 5.1 when less than eight layers of stretch film were applied, low seal integrity of wrapping (OR 172 at <10 s of seal integrity), higher dry‐matter content (OR 1.17–1.56 per % unit DM) and higher concentrations of acetic acid (OR 47.5 per g DM) and ethanol (OR 3.4 per g DM). Mycotoxin presence was not found to be correlated with any of the chemical characteristics of the baled forage, or with any forage production and management factors. A positive correlation was present between total fungal counts and general mycotoxin presence, but not between specific mycotoxins and the toxin‐producing fungal species.     

基因型与环境对小麦戊聚糖含量及RVA黏度的影响

为研究基因型与环境对小麦戊聚糖含量及RVA黏度的影响,选取10个小麦品种（系）和5个生态试验点,测定了小麦籽粒中的总戊聚糖(TP)、水溶性戊聚糖(WSP)、非水溶性戊聚糖含量(WIP)和RVA黏度等共10个性状,探讨了3种戊聚糖含量和7个RVA性状的影响因素及相互关系。结果表明,包括总戊聚糖、水溶性戊聚糖含量在内的10个性状均受到基因型、环境以及基因型与环境互作的影响。其中,基因型方差和环境方差在所有性状上均达到显著或极显著,基因型与环境互作方差在水溶性戊聚糖和7个RVA性状上达到极显著;戊聚糖含量的环境变异＞基因型变异＞基因型与环境互作变异,而RVA黏度性状的基因型变异均大于环境变异。水溶性戊聚糖含量与高峰黏度呈显著负相关。     

Comparison of palm and mixtures of refined palm and soybean oils on serum lipids and fecal fat and fatty acid excretions of adult humans

The effects of palm (P) and mixtures of palmand soybean (PS), palmolein and soybean (POS),palmstearin and soybean (PSS) oils on serum lipids andfecal fat and fatty acid excretions of humans werestudied. Each oil was the dominant fat in dietsconsumed by ten normolipidemic live-in individuals.Test diets were assigned by randomization. All fourdiets had a similar influence on total cholesterol (TC),low density lipoprotein (LDL), high densitylipoprotein (HDL), very low density lipoprotein(VLDL), triglycerides (TG) and phospholipids (PL). Fecalfat excretion was higher (1.81, 1.80 g/d) with PS andPSS and lower (1.78, 1.42 g/d) with P and POS diets.Excretions of palmitic (C16: 0), oleic (C18: 1), andlinoleic (C18: 2) acids were similar for all diets.Excretions of stearic acid (C18: 0) were higher whenfeeding POS and PSS, and lower with feeding P and PS;the changes were statistically significant atp < 0.05. More linolenic acid (C18: 3) was excretedwhen P was fed compared to the feeding of PS, POS, andPSS. Changes in linolenic acid excretion between P andPOS were statistically significant at p < 0.05. It wasconcluded that there were no significant differencesin serum lipid concentrations due to feeding palm orany of the refined palm and soybean oil mixtures.However, a significant difference was found in C18: 0excretions with feeding POS and PSS compared to whenP and PS were fed. Similarly, significant C18: 3excretion was found with feeding P compared to whenPOS was fed.     

The effectiveness of carbamate and copper compounds in the control of Cercospora beticola Sacc. and their effects on yield and quality composition of autumn sown sugar beets

The fungicidal effectiveness of Zn-carbamate and copper compounds and their effects on yield and quality composition of autumn-sown beets were investigated under field conditions in a subtropical area. The two kinds of compounds — at their specific optimal treatment combinations — were equally effective in their fungicidal action and in leaf preservation. Weekly applications were required in the warmer part of the growing season for optimal results; however, during the cooler, earlier period triweekly applications were sufficient. Sprinkler pre-irrigation was essential for effective control by the fungicide-treatments. Zn-carbamate significantly exceeded the two copper compounds in sucrose storage (by 1395 kg/ha). This was the result of a less phytotoxic — respiration enhancement — effect. Beaurdice, a metallic Cu-compound, exerted the most detrimental effect. The optimal treatment for sucrose storage increased: (a) the total sucrose by 5170 kg up to 18,430 kg/ha, (b) the daily net storage rate of sucrose by 311.4%, and (c) sucrose/m³ of water by 610–710 g. The effective treatments considerably improved the technological value of the beet by: (a) increasing the juice purity by 4.7% to 91.7%, (b) decreasing the sol. ash and sol. N ratios and (c) maintaining the sucrose concentration at the same, or slightly higher, level throughout the entire experimental season. It was shown that the Cu-ions restricted the sol. ash concentration in the beet by interfering with the uptake of the mineral elements or by enhancing their incorporation.     

Synthesis,characterization and ultrafiltration of reactive dyes. Application by exhaustion and/or ink-jet printing. IV.

Three new bis azo reactive dyes of different metallic salts (Na, K, Li) were synthesized. The synthesis was obtained by diazotization of 4-amino-2:5 di methoxy phenylene-beta hydroxyl ethyl sulphone sulphate ester and coupling with 1-amino-8-hydroxynaphthalene-3,6-disulfonic acid (H-acid) (moles ratio 2:1). The dyes were purified-concentrated by ultrafiltration technology, characterized and applied on cotton, wool and nylon 6,6 fabric by exhaustion (dyeing) and by ink-jet printing. Fastness properties of the dyeings were measured. Wash fastness was investigated according to international standard methods and was found to be very good to excellent in all cases, while light fastness values were medium to low. Ink-jet ink formulations were prepared using the ultra filtrated reactive blue dyes UF RB(a-c) and their properties pH, conductivity, surface tension and viscosity were monitored over a period of 90 days. The inks were used to print digitally cotton and nylon 6,6 samples and wash- and light fastness properties of the prints were measured. Wash fastness properties were excellent while light fastness values are low Colour measurements of the dyeings and prints were conducted.