一株石油烃降解菌TY22烷烃羟化酶基因的克隆及功能研究

[目的]对一株石油烃降解菌TY22的烷烃羟化酶基因alkB克隆测序,并对其外源表达和功能进行研究。[方法]PCR扩增获得alkB基因部分序列,再根据该序列通过染色体步移技术,最终获得完整CDS序列。将alkB基因与pET21b(+)载体构建重组质粒,并转入E.coli BL21(DE3)进行表达。再将alkB基因克隆至pCom8载体中,分别转入E.coli GEc137(p GEc47△B)和Pseudomonas fluorescens KOB2Δ1进行基因功能的互补试验。[结果]染色体步移获得了1 236 bp的烷烃羟化酶基因完整CDS序列(Gen Bank Accession:KU867870)。诱导表达发现,重组菌在0.1 mmol/L IPTG、30℃条件下诱导培养6 h的表达效果最佳,得到与预期相符的46 kDa蛋白。基因功能互补试验发现,重组菌能在以C12~C16为唯一碳源的培养基中生长。[结论]TY22菌株的烷烃羟化酶基因完整CDS序列全长1 236 bp,能氧化C12~C16的中长链烷烃,并能在大肠杆菌中有效表达。     

W/O微乳液体系中SA和MS清除·O_2~-的研究

以水溶液体系作为对照,利用分光光度法研究了W/O型微乳液体系中水杨酸（SA）和水杨酸甲酯（MS）清除超氧阴离子自由基（·O2-）的性能.结果发现,在水浴时间为20min、pH值为8.20和邻苯三酚加入量适宜的情况下,W/O微乳液体系中SA、MS对·O2-的清除率分别可达72.92%、81.47%.微小的pH值变化会引起邻苯三酚自氧化速率的显著变化,电解质溶液对其也有明显的影响,但对SA、MS清除·O2-的能力影响不大.     

普通烟草β-胡萝卜素羟化酶2基因克隆与生物信息学分析

β-胡萝卜素羟化酶(beta-carotene hydroxylase,BCH)是植物类胡萝卜素合成代谢中的关键限速酶,为获得烟草β-胡萝卜素羟化酶2(NtBCH2)基因序列,采用同源克隆法从烟草中分离NtBCH2的基因组DNA序列,基因登录号为JX101477。结果表明:1)同源克隆法从烟草中克隆出NtBCH2基因,NtBCH2基因组DNA全长2131bp,cDNA为1 083bp,含7个外显子和6个内含子。2)NtBCH2蛋白有309个氨基酸,分子量为34.79kD,具有7个糖基化位点,11个磷酸化位点,4个跨膜域。3)系统发育树与BLAST分析表明,NtBCH2是番茄的SlBCH和辣椒CaBCH2的同源基因;GENEVESTIGATOR数据库芯片结果表明,NtBCH2在幼嫩的茎和子叶中表达量最高。     

新型钴配合物为中性载体的水杨酸根电极研究

研究了基于水杨醛缩邻氨基苯酚合钴(Ⅱ)金属配合物[Co(Ⅱ)-SAAP]为中性载体的PVC膜阴离子电极。该电极对水杨酸根(Sal-)具有优良的电位响应性能和选择性并呈现出反Hofm e ister选择性行为,其选择性次序为Sal->C lO4->SCN->I->NO2->NO3->B r->C l->SO32->SO42-。在pH为5.0的磷酸盐缓冲体系中,电极电位呈现近能斯特响应,线性响应范围为9.0×10-6~1.0×10-1mol/L,斜率为-54.3 mV/dec(20℃),检出下限为5.0×10-6mol/L。采用交流阻抗技术和紫外可见光谱技术研究了电极的响应机理,结果表明配合物中心金属原子的结构以及载体本身的结构与电极的响应行为之间有非常密切的构效关系。该电极具有响应快、重现性好、检出限低、制备简单等优点。将电极用于药品分析,其结果令人满意。     

Molecular cloning and characterization of the cinnamate 4‐hydroxylase gene from Eupatorium adenophorum

Cinnamate‐4‐hydroxylase (CA4H), a cytochrome P450‐dependent monooxygenase, plays crucial roles in phenylpropanoid metabolism and plant defense. Previously, the authors showed that the expression of CA4H was induced in response to an allelopathic substance in Eupatorium adenophorum. Here, the full‐length cDNA of EaCA4H was cloned by using rapid amplification of cDNA ends. The 1518 bp open reading frame of EaCA4H was deduced to encode a protein of 505 amino acid residues. Like other CA4H proteins, the predicted EaCA4H polypeptides contained conserved domains of cytochrome P450. A Southern blot analysis indicated that at least five copies of EaCA4H exists in the genome of E. adenophorum. Subcellular localization revealed nuclear‐localized EaCA4H–green fluorescent protein fusion protein in onion epidermal cells. Heterologous silencing of endogenous CA4H in tobacco by a conserved EaCA4H fragment resulted in reduced expressions of key enzymatic genes and the production of downstream flavonoids in the phenylpropanoid pathway. Intriguingly, similar effects were observed in transgenic tobacco plants overexpressing EaCA4H. Altogether, the results indicate that the disturbed expression of CA4H in plants leads to relatively low expression levels of key enzymatic genes and the accumulation of the flavonoids that are involved in phenylpropanoid metabolism.     

NO3-N的水杨酸比色法快速测定

探讨了水杨酸比色法测定 NO3- N的基本原理与影响因素 ,确定了测定的线性范围 ,并用土壤及水体样品进行了标定。结果表明 ,1水杨酸比色法测定 NO- 3的吸收峰为 4 10～ 4 2 0 nm ,检出极限可达 0 .1mg/L ,比标准的酚二磺酸比色法灵敏 2～ 5倍 ;2水杨酸比色法测定 NO- 3,颜色稳定性强 ,加盖情况下可保存 15 d而无显著变化 ;3改进后的方法适应 p H范围大 (p H>2 ) ,可掩蔽高达 0 .2 m ol/L Cl-及 2 mg/L NO- 2 的干扰。研究认为 ,改进水杨酸比色法测定 NO3- N简便、快速 ,结果准确     

Tyrosine hydroxylase activity and dopamine turnover of rainbow trout (Oncorhynchus mykiss) brain: the special status of the hypothalamus

The dynamics of catecholamine (CA)-synthesis enzymes have been poorly studied in fish. Tyrosine hydroxylase (TH), the rate-limiting enzyme of CA synthesis has been only studied inin vitro conditions. In the present report thein vivo CA synthesis and the CA metabolism were studied in different regions of the forebrain of the rainbow trout. Levels of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) were determined by HPLC following a treatment with hydroxybenzylhydrazine (NSD), a potential inhibitor of DOPA decarboxylase. Kinetics of the accumulation of DOPA and of the decline of DOPAC were in agreement with those found in rat, evidencing that the accumulation of DOPA following NSD can be used in trout to quantify thein vivo enzymatic activity of tyrosine hydroxylase. Experiments using treatment with NSD or with methyl-p-tyrosine reached a same conclusion: the DA neuronal activity in trout is much higher than NE neuronal activity. However, the hypothalamus had high DA levelsvs. lowin vitro andin vivo TH activities and exhibited a low CA turnover.     

苯并_a_芘对大弹涂鱼肝脏芳烃羟化酶活性的影响

研究了苯并(a)芘(BaP)胁迫下大弹涂鱼肝脏芳烃羟化酶(AHH)活性的变化,结果显示BaP对大弹涂鱼肝脏AHH活性总体表现为显著的诱导作用(P＜0.01),其中BaP暴露的浓度及时间是影响AHH活性的重要因素.0.05mg@L-1浓度的BaP暴露对AHH活性无显著影响,而0.2mg@L-1和0.5mg@L-1浓度组AHH活性则显著被诱导(P＜0.01).随着暴污时间的延长,0.2mg@L-1浓度组AHH活性被显著诱导(P＜0.01),而0.5mg@L-1组AHH活性则相对稳定.对AHH活性变化的剂量-效应关系进行回归分析的结果表明,0.5mg@L-1浓度组暴露7d时,AHH活性受到一定程度的抑制.污染解除后,0.5mg@L-1浓度组AHH活性显著降低,恢复至与对照组相近,表明大弹涂鱼肝脏仍具有较强的生理调节机能,同时也表明AHH活性可及时反映环境中BaP的水平.以上这些结果表明AHH适于作为大弹涂鱼受BaP胁迫的生物指标.     

Methyl jasmonate and methyl salicylate, but not cis-jasmone, evoke defenses against infection of Arabidopsis thaliana by Orobanche aegyptiaca

The mechanism by which some plant species develop resistance to the root parasite, broomrape ( Orobanche aegyptiaca ), is still not clear. Resistance to other pathogens can be induced by methyl jasmonate and systemic acquired resistance can be induced by treatment with salicylic acid, while cis -jasmone can act as a signaling molecule in plant–insect interactions. The three compounds studied, methyl jasmonate, cis -jasmone, and methyl salicylate, were applied to Arabidopsis thaliana seedlings that were then transferred to Nunc cell culture plates and exposed to the germinating seeds of O. aegyptiaca . The number of infections of the roots of single seedlings of A. thaliana was then quantified. Exposure for 24 h to very low concentrations of methyl jasmonate or methyl salicylate, which were then removed, effectively induced resistance to infection of A. thaliana by O. aegyptiaca , reducing attachment and tubercle formation by 90%. cis -Jasmone was far less effective in inducing a similar resistance to infection. These results support the view that methyl jasmonate can induce almost full resistance to infection by broomrape. The fact that such resistance is not observed under normal conditions of infection supports the idea that the root parasite does not evoke the full defensive response in the host plant.