杜氏盐藻电击转化方法的系统优化

本研究系统分析了盐藻生长状态、电击条件、电击缓冲液成分和质粒浓度等条件对电击转化效率的影响。实验结果表明:正常接种后培养7d对数生长中期的盐藻细胞,在25μF、0.8kV的电击条件下加入终浓度为10μg/mL的质粒可使盐藻电击转化效率达到1.85‰;电击缓冲液中加入0.4mol/L的甘油可使转化效率显著提高至2.03‰(P<0.05)。在上述优化电击体系下,运用3种不同质粒分别转化盐藻细胞后获得的转化效率无显著差异。通过对电击转化中相关因素的优化,本研究建立了一种适用于杜氏盐藻的高效稳定的电击转化体系,为杜氏盐藻的转基因研究提供有效方法。     

高渗提高凝结芽孢杆菌P4-102B菌株的电击转化效率

【目的】凝结芽孢杆菌(Bacillus coagulans)是一种非常有工业应用前景的微生物,但遗传转化的困难是限制对其进行代谢工程改造的关键因素。本研究主要考察生长培养基、电击缓冲液、复苏培养基中添加高渗剂如山梨醇、甘露醇等对凝结芽孢杆菌转化效率稳定性的影响,并对凝结芽孢杆菌电击转化条件进行优化。【方法】用穿梭质粒p NW33N电转化凝结芽孢杆菌P4-102B,系统地考察高渗条件下细胞生长阶段、感受态菌体浓度、电击缓冲液组分和复苏培养基组成等因素对转化效率的影响。【结果】同一电场压力下高渗体系转化效率较低渗体系明显提高且稳定性较好,菌体在含有0.5 mol/L山梨醇的LB培养基中生长到OD600为0.8时收集,用SMG[0.5 mol/L山梨醇,0.5 mol/L甘露醇,10%(质量体积比)甘油]电击缓冲液洗涤菌体4次制备感受态,在固定电场强度14 k V/cm、脉冲时间5 ms、1 mm规格的电转杯进行电击转化,电转化后立即加入含有0.5 mol/L山梨醇和0.38 mol/L甘露醇的LB复苏培养基培养,能够获得最佳的转化效率2.7×102 CFU/μg DNA。【结论】使用高渗电击转化法能够提高电击转化的稳定性和重复性,并且可以获得较高的转化效率。     

杜氏盐藻质膜ATPase在渗透调节中的可能作用

杜氏盐藻是一种以甘油为渗透调节物质的单细胞海藻,能够在0.08～5.0mol/L NaGl的培养液中生长。当外界NaGl浓度从0.5mol/L上升到4.0mol/L时,藻细胞内的Na~+和K~+含量变化不大,甘油含量则从6.20Pg/cell上升到51.50pg/cell。当藻细胞承受2.0mol/L到3.0mol/L NaCl的高渗胁迫时,能通过增加细胞内甘油含量来恢复原有形态;同时,藻细胞的H~+分泌增加,ATP含量下降;20μmol/L Na_3VO_4抑制了这些变化。KGN处理虽降低藻细胞内的ATP含量,却增加K~+外流和Na~+内渗。     

南亚热带磷限制水库中浮游植物群落对氮磷比变化的响应

为了解P限制水体中浮游植物群落对N、P营养盐的响应,通过添加N、P营养盐设置N/P梯度,对广东省流溪河水库中的浮游植物群落进行了研究。结果表明,添加N、P显著促进浮游植物的生长,浮游植物群落受P盐的影响比N盐显著;藻类的种属特异性导致浮游植物群落对氮磷营养盐的响应不一致,浮游植物总丰度与N/P比值不相关,其中隐球藻(Aphanocapsa sp.)、拟柱胞藻(Cylindrospermopsis raciborskii)和假鱼腥藻(Pseudanabaena sp.)等蓝藻适合在高N高P条件下生长,双对栅藻(Scenedesmus bijuga)等绿藻优势种偏好中N高P环境,而曲壳藻(Achnanthes sp.)、小环藻(Cyclotella sp.)等硅藻在低N低P的环境下占据优势;P浓度为0.8～2.0μmol/L时存在诱导浮游植物碱性磷酸酶活性的阈值,当P浓度大于2.0μmol/L时则抑制酶活性; P浓度为2.0μmol/L可能是浮游植物维持生长的最适浓度,浮游植物N/P维持动态平衡;藻细胞N/P、C/P与水体P浓度、N/P呈显著正相关,而藻细胞C/N受N影响更明显(P0.05)。这为热带亚热带水库的水质管理提供了理论参考。     

锌对盐藻生长与物质积累的调控作用

实验研究了不同浓度的锌对盐藻细胞生长与物质积累的调控作用。结果表明,培养液中供给锌过多或过少都不利于盐藻细胞的生长与物质积累。以培养基中6 mg/L的锌浓度对盐藻细胞生长、蛋白质合成与β-胡萝卜素积累的促进作用最大。这一锌浓度可用于盐藻的生产性培养。当培养液中锌浓度较高(8 mg/L)或较低(2 mg/L)时,单个盐藻细胞中的蛋白质与β-胡萝卜素含量较高。但此时,因培养液中细胞密度较低,盐藻细胞积累的物质总量仍然较少。在锌浓度较高或较低的逆境条件下,盐藻可能通过适应性反应形成了逆境蛋白质与胡萝卜素等。     

磷酸盐对盐藻生长与物质积累的调控作用

实验研究了不同浓度的磷酸盐对盐藻细胞生长与物质积累的调控作用。结果表明,培养液中供给磷过多或过少都不利于盐藻细胞的生长与物质积累。以培养基中30mg/L的磷浓度对盐藻细胞生长、蛋白质合成与β-胡萝卜素积累的促进作用最大。培养液中磷浓度提高会使盐藻细胞生长与物质积累受到抑制。在培养液中的磷浓度为0mg/L时,单个盐藻细胞中的蛋白质含量最高。     

培养液NaCl浓度对杜氏盐藻3-磷酸甘油脱氢酶同工酶的影响

目的：探讨在不同NaCl浓度下,杜氏盐藻（Dunaliella salina）的3-磷酸甘油脱氢酶（Glycerol 3-phosphate dehydrogen-ase,GPDH）同工酶的活性与其渗透调节相关性。方法：采用聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis,PAGE）技术对在不同NaCl浓度生长的杜氏盐藻的GPDH进行同工酶电泳检测。结果：在0.5mol/LNaCl低盐的条件下,杜氏盐藻具有4种NAD＋-GPDH同工酶,分别为GPDH1、GPDH2、GPDH3、GPDH4。当NaCl逐渐分别增高为2.0、3.0、4.0、5.0mol/L时,只有1种NAD＋-GPDH同工酶即GPDH1。结论：GPDH1具有较高活性,这与高盐胁迫时细胞大量合成甘油进行渗透调节密切相关。     

C、N、P对杜氏盐藻突变藻株Zea1生长和积累玉米黄素的影响

通过UV辐照和NTG诱变处理杜氏盐藻野生型藻株得到一株杜氏盐藻高产玉米黄素突变株Zea1,以此突变藻株为实验材料,系统研究了光照强度、盐浓度、碳源、氮源、磷源等对Zea1生长和玉米黄素积累合成的影响。葡萄糖在10mmol/L时既适合Zea1生长,又有利于其玉米黄素的积累。KNO3浓度为1mmol/L时最适合突变株藻细胞生长,而(NH4)2SO4浓度为1mmol/L最有利于藻细胞内玉米黄素的积累。综合来看,1mmol/L(NH4)2SO4为最优氮源。KH2PO4浓度为0.1mmol/L时Zea1藻细胞积累玉米黄素含量最高,同时在此浓度下也最适宜藻细胞的生长。讨论了此结果的机理和意义,为利用杜氏盐藻大规模生产玉米黄素提供了理论依据。     

外源质粒电击转入Pseudomonas putida的条件研究

以自行筛选的恶臭假单胞菌（Pseudomonas putida）(命名为Rs198,Genbank登录号为FJ788425）为受体菌,将具有卡那霉素抗性标记的大肠杆菌假单胞菌穿梭质粒PDSK519通过电转化法导入到受体菌中,对细胞生长状态、电转化温度、质粒DNA及感受态细胞浓度、电击电压及电转化介质给予转化效率的影响进行研究。结果表明,在细胞生长至OD600为0.5左右时收集菌体,在低温条件下制备浓度为 4.6×1012/ml 的感受态细胞,以0.3mol/L的蔗糖为电转化介质,在13kV/cm的场强下电击能获得较高的转化效率,最高可达1.3×107个转化子/μ g DNA。为构建恶臭假单胞的遗传转化系统,利用基因工程手段为该菌的进一步研究奠定了理论基础。     

渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响

杜氏盐藻（Dunaliella salina）是一种抗渗透能力强的单细胞绿藻,甘油在其渗透调节过程中发挥重要作用。本实验对5种不同NaCl浓度条件下,盐藻的生长、细胞内甘油含量及甘油代谢相关酶的活性变化进行了测定。结果表明,NaCl浓度过高或过低均影响盐藻的生长;高渗胁迫条件下甘油含量迅速增加,3-磷酸甘油磷酸酶的活性和二羟丙酮还原酶催化二羟丙酮转化为甘油的活性明显增加;而低渗胁迫条件下的甘油含量会迅速降低,3-磷酸甘油磷酸酶的活性丧失,二羟丙酮还原酶催化甘油转化为二羟丙酮的活性增加。基于此实验结果,我们对盐藻渗透胁迫条件下细胞内的甘油代谢过程与其抗渗透胁迫能力的相关性进行了探讨。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.