特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

功能性新型冠状病毒RBD结构域在毕赤酵母表面的展示*

目的:设计并构建新型冠状病毒(SARS-CoV-2)受体结合结构域(receptor binding domain,RBD)在毕赤酵母表面的展示体系,并对表面展示的RBD进行功能性评价,从而为以RBD为靶点的高通量药物筛选平台奠定基础。方法:将四种锚定分子与新冠病毒RBD融合,电转化至毕赤酵母中;通过细胞免疫荧光分析,筛选能够成功展示RBD的锚定系统;进一步分析其与血管紧张素转化酶2(angiotensin-converting enzyme 2,ACE2)受体的亲和力,证明展示在细胞表面RBD分子的功能。结果:仅Sed1p锚定分子能够有效呈递RBD至毕赤酵母细胞表面,展示效率约为70%;亲和力分析结果表明,ACE2受体和表面展示RBD的亲和力(K_D = 30.42 nmol/L)与溶液中RBD的亲和力(K_D = 16.00 nmol/L)较为接近。结论:这一体系能够在毕赤酵母表面高效地展示具有生物学功能的RBD,可用于抗新冠病毒RBD药物的高通量筛选和评价。     

Thee effect of 5-HTP on the reproductive timer in the male cricket

The post-copulatory sexually refractory stage in the male cricket Gryllus bimaculatus consists of the two substages: the first refractory stage (RS1, time-variable) between copulation and spermatophore preparation, and the second refractory stage (RS2, time-constant) between spermatophore preparation and the recommencement of courtship. To understand the mechanism of the timer for RS2, subcuticular or intraganglionic injection of biogenic amines (10(-2) mol l(-1)) was performed immediately after spermatophore preparation. RS2 was shortened by octopamine, 5-HT, 5-HTP and NA-5-HT. Among these, 5-HTP was most potent. It shortened RS2 to maximally about 38% of the control. The shortening effect continued for 4.5 h after subcuticular injection even when the hemolymph was washed out with saline at 1 hour after injection. Simultaneous injection of 5-HTP with the inhibitor (NSD-1015) of 5-HT synthesis enzyme nullified the effect of 5-HTP, indicating that the shortening effect was caused by 5-HT synthesized from extrinsic 5-HTP. Injection of the inhibitor (CHX) of protein synthesis had no effect of on RS2. These results suggest that the reproductive timer in the TAG may be controlled by 5-HT or a second messenger mediated by 5-HT.     

五种丛枝菌根真菌对枳实生苗耐锌污染的影响

通过盆栽实验研究丛枝菌根(AM)真菌Glomus versiforme(G.v)、G.mosseae(G.m)、G.intraradices(G.i)、G.aggregatum(G.a)和G.etunicatum(G.e)在锌污染条件下枳实生苗的菌根侵染、生长、叶片和根系锌、磷含量及部分生理指标的影响.结果表明:锌污染...     

嗜酸氧化亚铁硫杆菌APS还原酶的表达、纯化及其性质鉴定

嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)中APS还原酶是硫同化途径的一个关键酶,其对硫酸盐的还原及硫化物的氧化具有重要调节作用.本文以A.ferrooxidans ATCC23270基因组为模板.通过PCR扩增得到编码APS还原酶的cysH基因,与原核表达载体pLM l构建重组体,转化入大肠杆茵(Escherichia coli,E.coli)DH5a中,测序正确后,加IPTG诱导表达,用一步亲和层析法纯化出浓度和纯度都较高的APS还原酶.由蛋白颜色和紫外分析,确定其含有一个[Fe4S4]簇作为活性中心.表达产物进行SDS-PAGE分析,证实分子量为28 kD.酶活测定表明其具有将APS还原为亚硫酸盐跟AMP的功能.     

黄山木兰的丛枝菌根定殖状况

在原产地和引种栽培地采集了国家珍稀濒危植物黄山木兰（Magnolia cylindrica）的根样及其根际土壤样品,研究了根中丛枝菌根真菌（AMF）的定殖状况。AMF在两地黄山木兰的根中均有定殖,并形成典型的Paris-type类型丛枝菌根(AM),原产地黄山木兰的AMF定殖率高于栽培地。用湿筛沉淀法从两地黄山木兰根际土壤中共分离鉴定出5属22种AMF,两地AMF群落相似性系数为0.96。原产地黄山木兰的AMF孢子密度高于栽培地,而Shannon-Wiener多样性指数低于栽培地。研究结果表明,黄山木兰引种栽培后并没有对其根际土壤中AMF的群落结构造成大的改变。     

Location of the reproductive timer in the male cricket Gryllus bimaculatus DeGeer as revealed by local cooling of the central nervous system.

The location of the reproductive timer for the post-copulatory, time-fixed, sexually refractory stage was investigated in the male cricket Gryllus bimaculatus. This stage was defined as the interval between spermatophore protrusion and recommencement of copulation or a calling song. To inactivate the central nervous system locally and reversibly, different body regions were cooled to 10 degrees C for 20-30 min after spermatophore protrusion. A behavioural test then measured the duration of the refractory stage after males recovered from cooling. Males with the head, thorax and anterior abdomen cooled did not show a lengthening of that stage. In contrast, males with the entire abdomen or even the posterior abdominal segments containing only the 6th and terminal (7th-11th) abdominal ganglia showed a lengthening of the refractory stage up to, but not exceeding, the cooling duration. When 20-min cooling was interposed twice after spermatophore protrusion, the refractory stage was lengthened by about 40 min, indicating that interposed cooling did not reset the timer. These results are in agreement with our previous hypothesis that the reproductive timer for the refractory stage in the male cricket is located in the posterior abdominal ganglia, possibly within the terminal abdominal ganglion.     

Serotonin precursor (5-hydroxytryptophan) has a profound effect on the post-copulatory time-fixed sexually refractory stage in the male cricket, <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis> DeGeer

This study addresses a potentially general basis of measuring time in a biological timer. Here, we examined the effects of biogenic amines on the time-fixed post-copulatory sexually refractory stage (ca. 1 h) which is defined as the time interval between spermatophore protrusion and the onset of a calling or a mating response in the reproductive cycle of the male cricket. For subcuticular injection of amines (0.15 ml, 10(-2) mol l(-1)), the interval of the refractory stage was shortened by octopamine, serotonin, 5-hydoxytryptophan and N-acetyl-serotonin but was unchanged by tryptophan, melatonin or 5-hydroxyindol-3-acetic acid. The effect of 5-hydoxytryptophan was most potent (maximum shortening, 38%) and long lasting (ca. 4.5 h) while other amines effected only the injected cycle. Injection of 5-hydoxytryptophan (180 nl, 10(-2) mol l(-1)) into the terminal abdominal ganglion also decreased the interval to a similar extent. Simultaneous injection of 5-hydoxytryptophan with the inhibitor of the serotonin synthesis enzyme reduced the 5-hydoxytryptophan effect suggesting that this effect results from synthesis of serotonin from 5-hydoxytryptophan. The protein synthesis inhibitor cycloheximide had no effect on the interval. These results suggest that the reproductive timer is regulated by serotonergic neurons in the terminal abdominal ganglion without protein synthesis during the interval of the time-fixed sexually refractory stage.     

十二烷基硫酸钠对斑马鱼抗氧化能力的影响

以斑马鱼Brachyclanio rerio为实验对象,用不同浓度(0.5、3.0、18.0mg/L)的阴离子表面活性剂十二烷基硫酸钠(SDS)进行3、14、28、42、63d的暴露实验,通过测定肌肉组织中的总抗氧化能力(TAC)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量三项指标研究SDS对斑马鱼抗氧化能力的影响。结果表明:0.5mg/L和3.0mg/L实验组SOD活性受到显著抑制(P<0.05),TAC水平和MDA含量与对照组无显著差异;18.0mg/L实验组在TAC水平和SOD活性上均表现为显著降低(P<0.05或P<0.01),MDA含量较对照和低剂量组亦有较大幅度增加,显示该组受试个体的抗氧化能力受到了显著抑制。研究结果对评价水环境中SDS毒性生物效应具有参考意义。