Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension- cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was dis- cussed.     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.     

三种ARS片段对水稻部分酶解小细胞团转化频率的影响

ＡＲＳ２,ＡＲＳ７,ＲＳ８是从水稻珍汕９７Ａ不育系统ＤＮＡ中的克隆的一组在酵母细胞中能直始ＤＮＡ复制的片段。其中ＡＲＳ８是单考贝而ＡＲＳ２,ＡＲＳ７却是高度重复顺序。将带有ＡＲＳ２,ＡＲＳ７,ＡＲＳ８的质粒用低压脉冲电泳法导入水稻部分酶解小细胞团中,经浅层培养,铺板,获得愈伤组织。     

Cu2＋猝灭茜素红荧光法检测谷胱甘肽

茜素红（ARS）与硼酸（H3BO3）之间能发生相互作用形成黄色配合物,使ARS的荧光增强,Cu2＋可以猝灭茜素红在Britton-Robison（BR）缓冲溶液中的荧光。加入谷胱甘肽（GSH）后,由于GSH所带的巯基（—SH）和Cu2＋发生络合,使体系的荧光强度增大,据此可以快速的测定GSH的含量。在pH 6.3的BR缓冲溶液中,茜素红-Cu2＋体系荧光强度的增强值与GSH的浓度在1.0×10-5～1.9×10-4 mol·L-1范围内呈线性关系,检测限为1.0×10-5 mol·L-1。     

双元载体提供组蛋白H3/H4拷贝1的elp3Δ菌株的构建

人Elongator是在转录中有功能的组蛋白乙酰转移酶(HAT)复合物,为研究其催化亚基Elp3功能,构建了酵母组蛋白H3/H4拷贝1的双元表达载体pRS316CFT,通过PCR介导的基因敲除,获酵母基因组H3/H4缺失由双元载体提供单拷贝H3/H4的elp3Δ菌株.敏感性实验表明该载体提供H3/H4可维持酵母正常生长.本工作为构建H3/H4乙酰化位点突变菌株,用功能互补在体内研究人Elp3 HAT活性功能奠定了基础.     

离子液体双水相中茜素红S与牛血清白蛋白作用研究

在离子液体四氟硼酸1-丁基-3-甲基咪唑(fBrnim]BF4)和NaH2PO4形成的双水相体系中,研究茜素红S(ARS)与牛血清白蛋白(BSA)生成复合物光谱行为和溶液酸度,盐浓度,离子液体用量,共存物质等对体系复合物测定影响.离子液体用量1.5 mL,磷酸二氢钠2.0 g,ARS 1.0 mL,蛋白质溶液1.0 mL,pH 6时ARS与BSA复合物吸光度较大.离子液相中复合物最大吸收波长位于531 nm,比单纯ARS红移109 nm;摩尔吸光系数(ε)为2.32×104L·mol-1·cm-1.用加入不同类型表面活性剂方法,初步探讨了ARS与BSA之间的作用机理.使用后的离子液体经过简单处理可以重复使用.     

刚毛柽柳TheIF1A基因转入酵母的抗逆能力分析

翻译起始因子eIF1A基因是蛋白合成的重要调控因子之一,在一些植物中可能参与抗逆调控过程。为了研究刚毛柽柳TheIF1A基因是否参与抗逆过程,将TheIF1A基因插入酵母表达载体pYES2中构建成重组载体,转入酿酒酵母(Saccharomyces cerevisiae)中获得重组型酵母,分别比较转TheIF1A基因酵母和转空载体对照酵母在山梨醇、H₂O₂、CdCl₂、CuSO₄、ZnCl₂、KCl、Na₂CO₃、MgCl₂、-20 ℃和53 ℃胁迫处理之后的存活能力。结果显示,TheIF1A基因能有效提高转基因酵母的抗干旱、盐碱、氧化、重金属及极端温度的能力,表明TheIF1A可能参与了柽柳多种抗逆调控过程。     

哌啶衍生物抗氧化作用结构-活性关系的理论阐释

哌啶衍生物(piperidine derivatives, 简称PDs)是一类新型抗氧化剂,具有较强的抑制脂质过氧化的能力.实验测得PDs的活性顺序为SH取代产物＞NH2取代产物＞OH基取代产物.本文用密度泛函理论(density function theory, 简称DFT)方法B3LYP/ 6-31G(d,p)计算了X-H(X＝O,N,S)键的解离焓(bond dissociation enthapies, 简称BDEs)以阐明PDs的结构-活性关系.结果表明,X-H BDEs的顺序是O-H＞N-H＞S-H.鉴于BDEs与抗氧化剂活性呈负相关,因此X-H BDEs的差异圆满解释了PDs的活性顺序,而前者又源于X原子电负性的差异,这提示实践中可以考虑用SH取代OH以进一步提高酚类抗氧化剂的活性.此外,本文还比较了半经验量子化学方法AM1和B3LYP/ 6-31G(d,p)对不同取代的PDs结构优化的效果,发现AM1方法对OH取代物的优化效果最好,对NH2,SH取代物优化效果较差.     

三种水份条件下湿生植物Lythrum salicaria L.的菌根状况及其对植物生长的影响

陆生植物的菌根已被广泛研究,对湿生植物菌根的研究却很少.本研究的目的是检测在水淹、潮湿和干旱条件下水生植物L.salicaria的菌根合成状况及其对植物生长的影响.在三种水份条件下,L.salicaria与来源于不同地点(干旱和水淹)的两种接种物(F2和F1)均可形成菌根,水份条件和接种物种类都显著地影响菌根的合成水平(P《0.05).F1和F2接种物与植物合成菌根的水平均在水淹处理中最低. F1接种物与L.salicaria合成菌根的水平在潮湿和干旱处理中显著地高于水淹处理(P《0.05),并且趋势是:潮湿》干旱》水淹处理.F2接种物与L.salicaria合成菌根的水平在三种水份处理中没有显著区别,但仍然可观察到一个趋势:干旱》潮湿》水淹处理.在水淹和干旱处理中,菌根对植物地上部分的生长和根的生物量积累没有影响.在潮湿处理中被F1接种的植株与被F2接种的植株与对照植株相比,地上部分干重、根鲜重显著地低;地上部分鲜/干重比、地上部分/根鲜重比显著地高.水份条件显著地影响植物的生长行为.     

红曲米中红曲霉的分纯及抗菌作用的研究

对广西武宣一带的红曲米中的红曲霉进行分离纯化.得到了8个纯培养.初步鉴定分属3类:M R 1,M R 2,M R 5,M R 6,M R 8属于M.ruber类(红色红曲菌);M R 3,M R 7属于M.purpureur类(紫色红曲菌);M R 4属于M.p ilosus类(丛毛红曲菌).以大肠杆菌(E.coli),枯草芽孢杆菌(B acillus subtilis),金黄色葡萄球菌(S taphy lococcus aureus),酿酒酵母(S accharomy ces serev isiae),青霉(P en icillium)为供试菌,运用滤纸片法对红曲霉培养液的抗菌效果进行检测.结果表明:红曲霉的培养液对细菌的抗菌效果明显,尤其是对革兰氏阳性菌-枯草芽孢杆菌抗菌能力较强;而对酵母和霉菌则无抗菌效果.获得了抗菌效果较好的3个红曲霉菌株作用.     

Increase of Expression Levels of Reporter Gene in Transgenic Tobaccos by Matrix Attachment Regions

The matrix attachment region (MAR) located downstream of Plastocyanin gene was isolated from the genome of pea. To study the effect of MARs on foreign gene expression in transgenic plants, T-DNA vector was constructed in which MARs flanked bothβ-glucuronidase(GUS) gene and selectable marker neomycin phosphotransferase (NPT-II) gene. The plant expression vectors were transferred into leaf discs via Agrobacterium-mediated transformation procedure. The result of GUS measurement showed that pea MAR could increase transgene expression level. The mean expression levels of GUS gene expression in population containing MARs could be increased twofold when compared with that of population without MARs.     

云南寻甸石漠化土壤易氧化碳对丛枝菌根真菌共生的响应

【目的】运用菌根技术改良石漠化土壤性状,已成为石漠化地区植被与土壤恢复的重要生物学途径。揭示丛枝菌根(AM)真菌与植物共生驱动下石漠化土壤碳库及养分状况的变化,探明石漠化土壤易氧化碳(ROC)对土壤碳库及养分变化的响应过程及机制,为石漠化土壤的微生物修复及提高石漠化治理效率提供参考。【方法】采集云南昆明寻甸石漠化土壤,以尼泊尔桤木(Alnus nepalensis)为寄主植物,分别接种摩西斗管囊霉(Funneliformis mosseae, FM)、幼套近明球囊霉(Claroideoglomus etunicatum, CE)、根内根孢囊霉(Rhizophagus intraradices, RI),并设置对照处理(无寄主植物及无AM接种),采用高锰酸钾氧化法测定不同试验处理下土壤ROC含量,探究土壤ROC与碳库组成、养分状况及植物生长之间的相互关系。【结果】不同AM真菌均具有显著的侵染与促生效应,其中RI的侵染率与菌丝侵染密度最大,相较于对照分别提高155%和100%,并显著促进桤木树高(60%)与基径(46%)生长;不同AM真菌均显著提高ROC含量,3菌种对ROC含量的提升率大小顺序为:RI(122%)> CE(78%)> FM(61%)。ROC在土壤总有机碳库中所占比例(52%)远高于微生物生物量碳(3%~6%);3种菌种对土壤养分含量的提升效应表现为:RI > CE > FM。相较于对照,RI 菌种对植物可利用性氮、微生物生物量碳、总有机碳及植物可利用性磷的提升率分别为161%、127%、110%及97%;对ROC变化具有较大贡献的土壤环境因子分别为AM真菌侵染率(96%)、植物可利用性氮(94%)、微生物生物量碳(85%)、总有机碳(78%)及植物可利用性磷(72%)。【结论】AM真菌与尼泊尔桤木共生,显著驱动石漠化土壤碳库与养分含量变化并促进植物生长,进而增加石漠化土壤易氧化碳的积累。研究结果有助于理解石漠化地区植物生长、土壤恢复及活性有机碳沉积的微生物学调控机制。     

中国人群DMD基因含外显子片段的限制性带型和RFLPs研究（Ⅰ）──BglⅡ片段的杂交带型及限制性片段长度多态的频率

采用ＤＭＤ基因的全长ＣＤＮＮ（４ｋｂ）为杂交探针，分析了中国人群ＤＭＤ基因含外显子片段（ｅｘｏｎ－ｃｏｎｔａｉｎｉｎｇｆｒａｇｍｅｎｔ）的ＢｇｌⅡ限制性片段和ＲＦＬＰｓ．新揭示出四个ＢｇｌＩＩ片段，ＤＭＤ基因全长范围内含外显子的ＢｇｌⅡ限制性片段总数至少为５９个，这为制作ＤＭＤ基因的ＢｇｌⅡ部分限制图奠定了重要基础；另分析了四个ＢｇｌⅡＲＦＬＰｓ在中国人群中的等位频率，在ｃＤＮＡ２ｂ－３、ｃＤＮＡ４－５ａ和ｃＤＮＡ５ｂ－７三个亚克隆探针的杂交带型中，杂合体频率预期值（ＥＨＦ）分别是０．３３、０．３３和０．４４，实际观察值为：０４０、０．４０、０．４８，表明有较高的临床应用价值．此外，在ＢｇｌⅡ／２ｂ－３和ＢｇｌⅡ／４－５ａ的带型中，还分别发现一个新的罕见等位片段．     

荒漠植物沙柳(Salix psammophila)根围AM真菌时空分布

为探明荒漠植物沙柳(Salix psammophila)根围AM真菌时空异质性,2009年5月、8月和10月分别从内蒙古荒漠植物沙柳根围分0～10,11～20,21～30,31～40和41～50 cm 5个土层采集土壤样品,研究了AM真菌时空分布及与土壤因子的相关性.结果表明,沙柳根围AM真菌平均孢子密度为282个/1...     

产肌酐水解酶菌株的分离、鉴定及产酶条件研究

研究分离到几株产肌酐水解酶的菌株,对所产酶的特性分析表明,菌株 42 1 表现较高的产酶性能且所产的酶具有高的热稳定性.对菌株42 1产酶发酵条件的研究表明,菌株 42 1 以酵母膏和玉米浆为氮源时表现较高的产酶性能,葡萄糖等容易利用的碳源的存在对酶的产生没有抑制作用.诱导物肌酐、肌酸、肌氨酸、氯化胆碱和尿素可以诱导酶的产生,Mn2+、Co2+对酶的产生有促进作用.     

Oxidative coupling of cinnamic acid derivatives and their radical-scavenging activities

4-Hydroxycinnamic acid derivatives, including ferulic acid (FA) (1), sinapic acid (SA) (2) and caffeic acid (CA) (3), are widely distributed in the plant kingdom, and undergo oxidative cross-coupling leading to the corresponding dehydrodimers, trimers and even higher oligomers in plants. In order to evaluate the antioxidative ability of these 4-hydroxycinnamic acid derivatives and their oligomers, we synthesized 8-8′-bis-lactone-dimers (8-8′-DiFA (4), 8-8′-DiSA (5) and 8-8′-DiCA (6)), as well as a new trimer (7), by the reaction of the corresponding monomers (1–3) with Ag2O in methanol, and assayed their free radical-scavenging activity by the reaction with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•). It was found that CA (3) and 8-8′-DiCA (6) bearing o-dihydroxyl groups exhibited significantly higher radical-scavenging activity than those bearing no such groups, and oxidative coupling of CA (3) resulted in remarkable enhancement in the activity.