丹参海藻糖-6-磷酸合成酶基因（SmTPS）的克隆及其表达分析

对丹参EST序列进行Blast分析,得到一条海藻糖-6-磷酸合成酶（TPS）基因,将其命名为SmTPS,GenBank注册号为JF937196。该基因cDNA全长2836 bp,包含一个长为2574 bp的ORF,编码857个氨基酸。生物信息学分析表明,分子量为97.04 kD,等电点为5.76,含α-螺旋、β转角、延伸连和无规则卷曲。该蛋白同时具有海藻糖-6-磷酸合成酶（TPS）和海藻糖-6-磷酸磷酸酯酶（TPP）的功能结构域。系统进化树分析表明丹参TPS功能域和拟南芥AtTPS6属于一类,而TPP功能域与低等生物酵母ScTPS2属于一类。实时定量PCR结果分析表明,SmTPS基因在丹参不同组织器官中差异表达,其茎中表达量最高。在干旱和低温处理下,其表达量与对照相比均可上调约2倍,表明SmTPS基因在丹参抵抗干旱和低温胁迫中发挥一定的作用。     

不结球白菜β-1,3-葡聚糖酶基因cDNA全长的克隆与表达分析

以不结球白菜(Brassicacampestrisssp.chinensis)抗病自交系雪克青为实验材料,通过RACE技术,获得了不结球白菜β-1,3-葡聚糖酶基因的cDNA全长序列。序列分析发现,该基因全长1275bp,编码363个氨基酸,分子量40.66kD,等电点(pI)9.27。推导的氨基酸序列与芜菁bg1基因具有96%的同源性,与拟南芥bg2基因具有61%的同源性,与其它植物β-1,3-葡聚糖酶基因的同源性为49%￣57%。将该序列提交GenBank,登录号为AY836001。Southern杂交显示该基因在不结球白菜基因组中的拷贝数多于1个。半定量RT-PCR分析表明,该基因有低水平的组成型表达,在霜霉病菌(Peronosporaparasitica)诱导后24h其表达量达到高峰。     

甘薯茎线虫β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫食道腺分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用.以马铃薯腐烂茎线虫(Ditylenchus destructor)为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b(GenBank登录号为FJ430142).此cDNA全长序列为1 640 bp,包括1个1 443 bp的完整ORF,编码1含481个氨基酸的蛋白,其理论分子量为50.89 kD,等电点pI为6.94.序列比对分析表明,含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBD Ⅱ).cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36、76、187和344bp,切割点符合5'-GT……AG-3'的规律.系统进化树分析表明,该基因与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测该基因可能来源于细菌的水平基因转移.     

白菜WRKY转录因子cDNA全长的克隆及分析

参照拟南芥WRKY转录因子基因保守序列设计引物，利用RT-PCR及RACE技术克隆了白菜WRKY转录因子基因cDNA全长，命名为BcWRKY1 (GenBank登录号为AY836002)。序列分析发现，白菜BcWRKY1基因核苷酸序列长980bp，推导的氨基酸序列编码285个氨基酸残基，与拟南芥AtWRKY18 氨基酸序列的同源性为74%，与拟南芥AtWRKY60同源性为59%；与其它植物WRKY基因的同源性则较低。Southern杂交显示该基因在白菜基因组中为单拷贝。半定量RT-PCR分析表明，用2mmol/L的水杨酸处理后2h~8h，以及用霜霉病菌接种后6h~12h，白菜BcWRKY1基因的表达量最高。     

甘薯肌醇-1-磷酸合成酶基因的克隆及序列分析

甘薯(Ipomoea batatas (L.) Lam.)新品系农大603是从感茎线虫(Ditylenchus destructor)病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。以农大603和徐薯18块根的mRNA为模板，根据植物抗线虫病基因NBS保守氨基酸序列设计引物，进行 RT-PCR分析，发现农大603的肌醇-1-磷酸合成酶(Myo inositol-1-phosphate synthase , MIPS)基因的表达量高于徐薯18。采用3'RACE技术扩增出MIPS基因的3'末端cDNA。根据植物MIPS基因 5'端一段保守的氨基酸序列设计兼并引物，并与3'端的特异引物组合，扩增出该基因的5'端cDNA序列。DNA序列比对表明，甘薯MIPS基因与大豆(Glycine max)、番茄(Lycopersicon esculentum )的MIPS基因同源性较高，分别达83.63 %和83.89 %。甘薯MIPS基因全长cDNA的克隆，有利于进一步研究该基因与抗甘薯茎线虫病的关系。     

猪多潜能性维持因子5 cDNA全序列的克隆与分析

利用前期筛选出的一些与母猪妊娠相关的有价值的新候选基因,选取其中一个位于网络顶端的EST序列(CK455976),根据此EST序列设计引物,以母猪卵巢为材料提取总RNA,运用RT-PCR和RACE-PCR对其进行cDNA全序列克隆,得到多潜能性维持因子5(developmental pluripotency associated 5,DPPA5)基因cDNA全长596 bp,提交GenBank数据库,登录号为FJ436413.经序列分析发现,其中5'UTR长67 bp,3'UTR长175 bp,编码区长354 bp,编码117个氨基酸.同源性比对分析发现,该基因核苷酸序列与人、小鼠、猕猴和狗的DPPA5基因同源性分别为83%、70%、83%和80%;编码的氨基酸序列同源性分别为74%、55%、74%和69%.经生物信息学分析,预测出DPPA5的相对分子量约13.60 kD,等电点5.24,在约1～10、18～29、38～51、60～66和75～110位之间氨基酸存在亲水性区域,DPPA5蛋白主要存在于细胞膜外侧.     

甘薯茎线虫(Ditylenchus destructor)β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫口针分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用。本文以甘薯茎线虫为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b (GenBank登录号为FJ430142)。此cDNA全长序列为1640bp,包括一个1443bp的完整ORF,编码一含481个氨基酸的蛋白,其理论分子量与等电点分别为50.89KD,pI为6.94。序列比对分析表明含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBDⅡ)。cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36bp、76bp、187bp和344bp,切割点符合5‘-GT...AG-3’的规律。系统进化树分析表明,该基因与细菌 Bacillus.subtilis和Erwina carotovora 分泌的纤维素酶属于同一支。     

甜瓜属野生种铝胁迫诱导基因ChAI的克隆及序列分析

应用同源序列克隆策略从铝盐胁迫处理的甜瓜属野生种(Cucumis hystrix Chakr.) 叶片中获得了612bp的片段,通过在EST数据库进行同源检索,发现一条甜瓜EST序列（GenBank登录号：AM724963.2）与之高度同源,据此设计引物并经RT-PCR扩增和序列拼接,最后获得了铝诱导基因cDNA序列全长,命名为ChAI (GenBank登录号：FJ968438 )。序列分析表明该基因全长967bp,其中开放读码框(ORF)长711bp编码236个氨基酸组成的多肽,5′ 和3′ 端非编码区长度各为15bp和241bp。DNAMAN同源性分析表明该基因与葡萄、棉花、羊乳（轮叶党参）、白骨壤（真红树植物）、拟南芥等植物的铝诱导蛋白的氨基酸序列具有78.6%以上的同源性。运用半定量RT-PCR技术对ChAI 基因的转录水平进行分析,根据该基因在铝胁迫下大量表达推测ChAI 与耐铝盐胁迫响应相关,但具体耐铝胁迫机制还有待深入研究。     

早酥梨抗黑星病相关新基因Vnp1的克隆及其表达分析

在前期已克隆得到的早酥梨(Pyrus bretschneideri cv.Zaosu)抗病基因同源类似物Pb-Zs3(GenBank登录号:EU939783)的基础上,通过RT-PCR获得Pb-Zs3的cDNA序列,并利用RACE技术获得该基因的全长cDNA序列,命名为早酥梨抗黑星病相关基因(Vnp1),提交至GenBank(GenBank登录号:FJ763188).生物信息学分析表明,早酥梨Vnp1基因的全长cDNA序列为1200 bp,包含一个完整的921 bp的开放读码框架(ORF),编码306个氨基酸残基,预测其分子质量为34.6 kD.同时,该基因含有与抗病相关的功能结构域核酸结合位点(NB-ARC),同源性比对显示其与苹果黑星病菌(Venturia inaequails)诱导后的苹果EST序列ABEA004598(GenBank登录号:EB150292)同源性达71%.对早酥梨Vnp1基因进行半定量RT-PCR研究的结果表明,该基因在梨黑星病菌(Venturia nashicola)诱导后216 h内的叶片中的表达水平呈明显上升趋势.     

白菜NAC转录因子BrcCUC3基因克隆及其对叶缘和主枝发育的影响

植物NAC (矮牵牛NAM基因、拟南芥ATAF1/2和CUC2基因)转录因子CUP-SHAPED COTYLEDON(CUC)亚家族成员在植物茎尖分生组织形态建成、器官边界分离、叶发育方面发挥着重要作用.采用基因同源克隆的方法获得了白菜(Brassica rapa ssp.chinensis)基因BrcCUC3,并转化拟南芥(Arabidopsis thaliana)做了初步的功能鉴定.研究结果表明,白菜Brc CUC3的编码区长1 008 bp,编码335个氨基酸,基因结构分析显示BrcCUC3包含3个外显子、2个内含子,内含子剪接位点符合GT-AG规则.氨基酸序列分析表明,BrcCUC3蛋白具有典型的植物NAC domain结构域.BrcCUC3编码区氨基酸序列与其它植物的CUC3蛋白有很高的一致性,尤其与甘蓝(Brasica oleracea)、萝卜(Raphanus sativus)和拟南芥CUC3蛋白高度一致,一致性分别达到98％,97％和83％.在不同物种CUC3的系统进化树上,BrcCUC3归属于双子叶植物分支的十字花科亚组,由不同植物20条CUC3编码区氨基酸序列所建立的系统进化树与真实的植物进化基本一致.荧光定量PCR分析结果表明,BrcCUC3在白菜叶深裂株系叶片中的表达量比叶全缘叶片中的高.利用根癌农杆菌(A grobacterium tumefaciens)浸花法转化拟南芥,获得了转BrcCUC3基因的拟南芥植株.过表达BrcCUC3的转基因拟南芥呈现叶缘出现裂刻、主枝增加的新表型.初步说明该基因参与叶形和主枝的发育调控,为揭示白菜叶形发育分子调控机制和通过基因工程创制植物叶形新种质提供分子依据.     

Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2)

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins.     

Excretion of (14)C-fumonisin B(1), (14)C-hydrolyzed fumonisin B(1), and (14)C-fumonisin B(1)-fructose in rats.

14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form.     

Chemoenzymatic synthesis of homochiral (R)- and (S)-karahanaenol from (R)-limonene

Terpinolene oxide, a monoterpene belonging to the p-menthane group, is easily derived from naturally abundant (R)-limonene. It was isomerized with montmorillonite clay catalyst to karahanaenone (2,2, 5-trimethylcyclohept-4-en-1-one) by ring enlargement. The enantiomers of the corresponding alcohol, karahanaenol (2,2, 5-trimethylcyclohept-4-en-1- ol), known for their individual organoleptic properties, were resolved through Pseudomonas cepacia lipase mediated enantiospecific alcoholysis of its acetate derivative.     

(三唑基-~(14)C-)粉锈宁的标记合成

本文报道了(三唑基-14C)-粉锈宁的制备。由14C-甲酸和重碳酸氨基胍形成(5-14C)-3-氨基-1,2,4-三唑,再经重氮化脱氨得到(5-14C)-1,2,4-三唑,最后再与对氯酚和二氯片呐酮反应得到(三唑基-14C)-粉锈宁。放化收率为26％(从甲酸-14C计),放化纯度大于95％。     

Theoretical speciation of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) in agronomic conditions

The presence of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) as the second largest component in commercial EDDHA iron chelates has recently been demonstrated. Here is reported the speciation of o,p-EDDHA by the application of a novel methodology through the determination of the complexing capacity, protonation, and Ca(2+), Mg(2+), Cu(2+), and Fe(3+) stability constants. The pM values and species distribution in solution, hydroponic, and soil conditions were obtained. Due to the para position of one phenol group in o,p-EDDHA, the protonation constants and Ca and Mg stability constants have different values from those of o,o-EDDHA and p,p-EDDHA regioisomers. o,p-EDDHA/Fe(3+) stability constants are higher than those of EDTA/Fe(3+) but lower than those of o,o-EDDHA/Fe(3+). The sequence obtained for pFe is o,o-EDDHA/Fe(3+) >/= o,p-EDDHA/Fe(3+) > EDTA/Fe(3+). o,p-EDDHA/Fe(3+) can be used as an iron chelate in hydroponic conditions. Also, it can be used in soils with limited Cu availability.     

Binding studies of oxovanadium(V) with ovalbumins (OA)

The effect of protein oxovanadium(V) ion concentration and pH on the ratio of diffusion current (id/id₀) was studied in vanadium(V) ovalbumin-S and denatured ovalbumin systems. In both the cases marked decrease in diffusion current was observed at the respective pH values, indicating that binding takes place with cationic groups of the proteins. The binding sites (n) were found to be pH dependent. The uniformity of logK and ΔG ⁰ value at all pH values indicated the involvement of same sites in interaction. Furthermore, the linear scatchard plots in both the systems supported the involvement of single class of independent sites in oxovanadium(V) anion interaction. The difference in binding sites (n) has been attributed to the folded structure of ovalbumin-S while unfolded one of denatured ovalbumin.     

Effect of pH on interaction between As(III) or As(V) and manganese(IV) oxide

The efficiency of As(III) oxidation by MnO₂, and retention of oxidation products varies with system pH. Maximum retention by hydrous Mn(IV) oxide occurs at pH < 5, declining at higher pH to about half total As at pH 10. The adsorption capacities of pyrolusite and cryptomelane at pH ～6.5 for As(V) species were 10 and 25 mmol kg^?1, respectively. HMO surface saturation (～10 mmol kg^?1) was reached with equilibrium As(V) levels of 5 to 8 × 10^?6 M but this was supplemented at higher levels by an absorption process where uptake increased linearly with concentration (e.g., 68 mmol kg^?1 with 2 × 10^?5 M As(V)). Added As(III) was avidly oxidized and most product retained at pH 3. At higher pH increasing amounts of As(III) remained unoxidized due to initial reactions apparently blocking access to internal pores. Added Na⁺ reduced the amount of As retained by the HMO, with the phosphate salt having a significant effect. Extraction studies confirmed that most As could be released by exposure to reducing agents or chelating agents (EDTA). The environmental significance of the results has been considered.