带内含子卡那霉素抗性基因双元载体构建及烟草转化

农杆菌介导法是植物基因转化的常用方法,然而由于筛选培养基中常用的抗生素头孢霉素和羧苄青霉素具有类植物激素活性,影响外植体的再生和转化频率。将一个植物的内含子插入卡那霉素抗性基因编码区的N端,合成了一个带内含子的卡那霉素抗性基因。构建带该基因的植物双元表达栽体pYP1202并转化烟草,受侵外植体在含卡那霉素50～200mg/L的选择培养基中抗性芽分化频率不受卡那霉素浓度影响,然而具有GUS活性的转化子占分化芽的比例却随着卡那霉素浓度的增加而升高。当培养基中加入500mg/L羧苄青霉素后受侵外植体产生的抗性芽频率比单一的卡那霉素筛选提高近1倍,高达91.4％,然而具GUS活性的转化子占抗性芽的比例仅有26.7％,在200m/L的卡那霉素筛选下,比例升至93.3％。用带内含子卡那霉素抗性基因构建的植物表达载体转化植物可以减少假抗性芽的产生。     

长效促胰岛素降糖酵母的构建及其对糖尿病模型小鼠的治疗效果

益生菌生物药物是指通过口服表达药用多肽(蛋白)的重组益生菌活细胞达到治疗疾病的新型口服给药系统。为了构建一种能有效防治2型糖尿病的酵母生物药物,文章首先构建了酿酒酵母(S.cerevisiae)整合型表达载体pNK1-PGK,并且通过绿色荧光蛋白(GFP)证明其表达功能正常,利用该载体将10×GLP-1 (Glucagon-like peptide-1)基因转化到酿酒酵母INVSc1中,通过营养缺陷型和Western blotting成功筛选出表达10×GLP-1的长效促胰岛素降糖酵母(Long-acting GLP-1 hypoglycemic yeast, LHY)。该酵母生长迅速,外源基因10×GLP-1表达稳定,表达量达到1.56 mg/g细胞湿重。通过链脲佐菌素和高脂高糖饮食联合诱导的方法构建了2型糖尿病小鼠模型,用LHY对其进行口服灌胃治疗,证明LHY具有较好疗效,明显降低血糖水平。     

非生物胁迫下植物表观遗传变异的研究进展

植物在整个生命过程中固着生长,不能主动躲避外界不良环境的危害,需要通过自身的防御机制来抵御和适应外界胁迫,而表观遗传修饰在调控植物应对不良环境胁迫中起重要作用。该文从DNA甲基化、组蛋白修饰、染色质重塑和非编码RNA等方面进行了综述,主要阐述了近年来国内外有关非生物胁迫下植物的表观遗传变化,以期为利用表观遗传变异提高植物的抗胁迫能力提供参考。     

无芒雀麦愈伤组织在氩离子（Ar^＋）注入过程中冻害耐受性研究

以无芒雀麦愈伤组织为研究对象,探讨了甘油处理对愈伤组织在氩离子注入过程中冻害的防护.结果表明:经甘油处理后,当离子注入剂量小于2.6×1015 Ar+·cm-2时,愈伤组织存活率在85%以上,失水率在12%,比未经甘油保护的有大幅度提高,甘油预处理的愈伤组织生长比较正常.注入剂量增加到一定程度,愈伤组织不能继续生长.     

昆虫神经生物学研究技术:触角电位图记录

触角电位图记录是昆虫神经生物学中用于昆虫嗅觉、信息素生物测定研究的重要技术之一。该技术具有敏感性、选择性高及操作简单易行等优点。该文以荷兰Syntech公司的产品为例详细介绍了此技术原理、装置、操作及应用     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins

The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low μM K(i) values in vitro. Their potential in vivo effects should be further studied.     

盐胁迫对三角叶滨藜根选择透性和反射系数的影响

以Hoagland溶液培养的三角叶滨藜幼苗为材料,分别采用电导率法、原子吸收分光光度计法和压力室法测定了不同浓度盐胁迫下三角叶滨藜根的质膜透性、离子吸收和根系反射系数,分析其抗盐特点和机制.结果表明:随着盐胁迫强度的增加,三角叶滨藜根细胞质膜透性增大、根系反射系数减小;盐胁迫导致三角叶滨藜根系对K+的总吸收量减少、对Na+的总吸收量增多,但对Na+的相对吸收量减少、对K+的相对吸收量增加.盐胁迫条件下,三角叶滨藜根系对离子吸收有较强的调节能力;而根系反射系数的减小有利于根系用较小的负压力吸收水分,减小木质部空化的危险.说明三角叶滨藜具有较高的抗盐能力.