杨树SAMT基因超表达载体的构建及遗传转化

一年生草本植物受到病原菌侵染时,体内的水杨酸甲基转移酶基因(salicylic acid methyltransferase,SAMT)能将植物侵染部位产生的水杨酸(salicylic acid,SA)转化为水杨酸甲酯(methyl salicylate,MeSA),在SA信号转导和植物系统获得抗性(systemic acquired resistance,SAR)中起着重要作用.而多年生木本植物中SAMT基因的功能还有待进一步验证.本研究根据毛果杨(Populus trichocarpa)SAMT基因序列,设计了84K杨SAMT引物,利用Gateway克隆技术,在BP反应酶作用下,使带attB接头的SAMT基因全长开放阅读框与入门载体pDONRTM222进行BP重组反应,将目的基因转入入门载体;转化大肠杆菌(Escherichia coli)感受态细胞DH5α后提取的质粒用MLUI进行酶切,然后在LR酶作用下与超表达载体pMDC32进行LR重组反应,将SAMT转入表达载体,成功构建了SAMT的超表达载体.通过农杆菌(Agrobacterium tumefaciens)介导的遗传转化,获得了SAMT超表达的84K杨转基因植株.为今后研究该基因在杨树抗溃疡病中的功能以及杨树在获得SAR和其他方面的作用提供了基础资料,同时也为研究该基因在其他多年生木本植物中的功能提供参考.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.     

木麻黄树皮提取物的清除羟自由基活性

考察了4种木麻黄树皮水提取物的化学组成及其对绝对分子质量分布的影响因素,用邻二氮菲-Fe2+氧化法测定了不同树种和不同溶剂提取物对羟自由基的清除率.结果表明,不同提取溶剂对木麻黄树皮提取物组成、绝对分子质量分布影响较大.盐酸-丙酮-水溶液(HAW,1.0%盐酸与丙酮溶液质量比7∶3)作提取溶剂可大幅提高提取物的总固形物和多酚含量.应用激光散射-凝胶渗透色谱(SEC)技术测得山地木麻黄树皮HAW提取物的绝对分子质量为5×102～2×105.木麻黄提取物清除羟自由基的活性高,质量浓度为2g/L的山地木麻黄树皮HAW提取物对羟自由基的清除率高达81.6%.提取物的组成影响其清除羟自由基活性.     

海洋活性胶原肽酶解液的脱色脱腥工艺

以富含胶原的海产品下脚料为原料,用海洋碱性蛋白酶894水解,得到具有清除羟自由基活性的酶解液.采用一次正交回归实验设计及结果分析,建立回归方程,研究了活性炭、β-环糊精、酵母三者用量及温度、pH和作用时间6因子及其可能存在交互作用的变化关系对该酶解液综合脱色脱腥效果的影响.结果表明,在给定取值范围内,pH对综合脱色脱腥效果影响高度显著,酵母添加量、pH与酵母添加量的交互作用影响显著,其它因子及其可能的交互作用影响不显著,得最佳工艺为:温度37℃、pH 4.0、活性炭0.8%、β-环糊精和酵母分别为0.1%,作用30 min.此时,综合脱色脱腥效果值为90.90,蛋白回收率为98.02%,羟自由基清除率的IC50浓度从原来的1.42 mg·mL-1增加到1.62 mg·mL-1.     

Sonme Factors that Affect the Free Radical-scavenging Activity of Tea Extracts

Some factors that affect the free radical-scavenging activety of two tea extracts were studied in vitro.It was found that concentration of tea extract or heating tea extract or treating with activated carbon and diatomite all had obvious effect on the scavenging activety of green tea extract,but heating or treating with diaomite had less effect on the scavenging activity of black tea extract.Ascorbic acid,for having synergic effect with tea extracts,could enhance the scavenging activity of tea extracts markedly,and the contrary was cupric ion.Reducing sugars such as fructose and glucose also had some syncrgic effect to tea extracts.     

Effect of SerpinE2 on metabolism of extracellular matrix

Serpin peptidase inhibitor clade E member 2 (SerpinE2), a member of serine peptidase inhibitor super family, regulates multiple serine proteases. Extracellular matrix (ECM) plays an important role in stem cell differentiation, signal transduction, tumor metastasis, osteoarthritis physiological and pathological processes and so on. SerpinE2 regulates the protein of extracellular matrix by serine protease system and matrix metalloproteinase (MMP) system. In this paper, we elaborate the effect of SerpinE2 on metabolism of extracellular matrix, and it will provide new ideas to find new therapeutic targets.     

华木莲叶黄酮类化合物抗氧化作用

通过邻苯三酚自氧化法,水杨酸比色法分别测定了华木莲叶黄酮类化合物(Sinomanglietia glaulaLeaf F la-vonoids,SLF)清除超氧阴离子自由基(O2.)、羟基自由基(.OH)的效果,及其对H2O2诱导的大鼠血红细胞溶血作用的影响来研究SLF的抗氧化性作用。结果表明:不同浓度的SLF均能显著地清除超氧阴离子自由基(O2.)、羟基自由基(.OH),抑制H2O2对红细胞膜的破坏而引起的溶血,抗氧化性作用随SLF浓度的升高而增强。     

棉花甲基化酶基因COMT和CCoAOMT的组织特异性表达分析

本研究利用半定量RT-PCR分析方法探索了陆地棉(Gossypium hirsutum L.)木质素合成途径中甲基化酶基因家族COMT和CCoAOMT在不同组织和不同发育时期纤维中的表达情况。结果表明COMT1,COMT2,COMT3和CCoAOMT1,CCoAOMT2在棉花的7个组织器官(根、茎、叶、子叶、花瓣、雄蕊、胚珠)中都有表达,其中COMT1和CCoAOMT1在各组织中的表达趋势类似,COMT2,COMT3和CCoAOMT2表达趋势各异。在5～25d的棉纤维发育过程中,COMT1和COMT3表达稳定,COMT2表达量逐渐增加。而CCoAOMT基因家族的2个基因的表达量变化相对明显,CCoAOMT1在25d出现表达高峰,CCoAOMT2则在10d和15d呈现高表达量。本研究表明甲基化酶基因家族COMT和CCoAOMT在棉花的根,茎等维管组织中具有相对一致的高表达量,而在其他组织和发育纤维中则呈现出明显的组织特异性和纤维发育时期特异性。     

大叶羊蹄甲总黄酮的提取及其对羟自由基的清除作用研究

为了研究大叶羊蹄甲中总黄酮的提取、鉴别方法及对羟自由基的清除作用。用乙醇提取法提取大叶羊蹄甲中黄酮类物质,采用分光光度法对提取物中总黄酮含量及羟自由基清除能力进行测定。提取大叶羊蹄甲中总黄酮的最佳工艺条件为：回流温度为80℃,乙醇体积分数为80%,固液比1:20,提取时间为1 h,在此条件下,黄酮的提取率为11.81%。此方法实验步骤简便,无污染,是提取大叶羊蹄甲中黄酮类化合物的有效途径,大叶羊蹄甲中总黄酮提取液对Fenton体系产生的?OH自由基有很好的清除作用。