人肝金属硫蛋白突变体β基因通过同源重组在蓝藻中的克隆与表达

将人肝金属硫蛋白（ＭＴ）突变体β基因插入到中间载体ｐＲＬ－４３９上的强启动子ＰｐｓｂＡ 下游,利用载体ｐＲＬ－β上的ＰｐｓｂＡ 和β基因与ｐｈａｓｍ ｉｄ ｐＴＺ１８－８上整合平台ＰｓｂＢ,构建整合表达载体ｐＴＺ－β．整合平台ＰｓｂＢ与集胞藻（Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３）染色体ＤＮＡ 上ｐｓｂＢ基因下游片段为同源序列．为了发生单交换同源重组,将外源基因β插入到整合平台ＰｓｂＢ下游的克隆位点．利用自然转化方法将表达载体ｐＴＺ－β整合到Ｓｙｎｅｃｈｏｃｙｓｔｉｔｓｐ．ＰＣＣ６８０３的染色体上．经氨苄青霉素筛选得到遗传性状稳定的转基因蓝藻．Ｓｏｕｔｈｅｒｎ ｂｌｏｔｔｉｎｇ 证明β基因已整合到Ｓｙｎｅｃｈｏｃｙｓｔｉｓ ｓｐ．ＰＣＣ６８０３的染色体上;Ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ 表明β基因已在蓝藻中表达．ＥＬＩＳＡ 测定在Ｚｎ２＋ 浓度为１５０ μｍ ｌ／Ｌ时表达量最高,为５９０ μｇ／ｇ 鲜藻;原子吸收表明转β基因的藻对Ｚｎ２＋ 的富集能力约为野生型的２倍．     

抗真菌蛋白Rs—AFPs基因在大肠杆菌中的表达

将抗真菌蛋白Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２全长ｃＤＮＡ插入表达质粒ｐＥＴ－２２ｂ／ＮｃｏＩ＋ＳａｃＩ位点,构建成融合蛋白表达载体ｐＲＡＦ１和ｐＲＡＦ２．将不含信号肽编码序列的Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２ｃＤＮＡ分别插入ｐＥＴ－２２ｂ／Ｎｃｏｌ＋Ｓａｃｌ和ｐＥＴ－２２ｂ／Ｎｄｅｌ＋ＳａｃＩ位点,构建成不含信号肽序列的融合蛋白表达载体ｐＲＡＦ３、ｐＲＡＦ４和非融合蛋白表达载体ｐＲＡＦ５和ｐＲＡＦ６．将构建的上述各种表达载体转化Ｅ．ｃｏｌｉＢＬ２１,挑菌落培养,ＩＰＴＧ诱导,使Ｒｓ－ＡＦＰｓ基因得到表达,并用体外抑菌试验检测表达产物的活性,结果表明,各种表达载体的表达产物均具有不同程度的抑菌活性,其中,ｐＲＡＦ３和ｐＲＡＦ４表达产物的抑菌活性较明显．     

cIts857基因的克隆、修饰及温敏诱导表达载体

从噬菌体λＤＮＡ（ｃＩｉｎｄｌｔｓ８５７Ｓａｍ７）克隆出９９０ｂＰ的ｃＩｔｓ８５７基因,经缺失,点突变等修饰改造和ＤＮＡ序列测定,获得了带有自身启动子区的修饰型ｃＩｔｓ８５７基因（７７０ｂｐ）。进而利用它构建大肠杆菌表达载体ｐＢＬＭＶＬ２和一套含３种不同阅读框架ｌｉｎｋｅｒ区的表达载体ｐＢＬＭＦＶ４、５Ｂ、６。上述表达载体;用于表达人工合成的牛生长激素（ｂＧＨ）、人干扰素－γΔＣ－１３和酵母ｐｈｏ８５等外源基因,都观察到这些基因产物的温敏表达。这些表明,克隆、修饰并组建到表达载体中的λ噬菌体ｃＩｔｓ８５７基因表达出具有功能作用的转录阻遏蛋白,使上述ＰＬ启动子控制下的大肠杆菌表达载体可经温敏诱导而高效表达外源基因产物．     

苏云金芽孢杆菌(Bt)晶体毒蛋白基因在烟草叶绿体中的表达

将全长３．５ｋｂ的Ｂｔ基因３’端缺失,得到长为２．１ｋｂ、１．８ｋｂ的基因。分别将这３个长度（１．８ｋｂ、２．１ｋｂ、３．５ｋｂ）的基因置于水稻叶绿体ｐｓｂＡ基因的启动子和终止子调控之下,并与选择标记基因ａａｄＡ（编码氨基糖苷－３’－腺苷酸转移酶,具壮观霉素抗性）表达盒相连;以烟草叶绿体基因ｔｒｎＨ－ｐｓｂＡ－ｔｒｎＫ为同源片段,构建成叶绿体转化载体ｐＢＴ３、ｐＢＴ８和ｐＢＴ２２。用基因枪把Ｂｔ基     

多能硫杆菌RbisCO基因在大肠杆菌中表达产物分析

通过对多能硫杆菌ＲｕｂｉｓＣＯ的基因表达分析表明该基因能够在ｐＢＲ３２２的Ｐ１启动子、ｐｕＣ１９的ｌａｃ启动子以及ｐＫＫ２２３－３的ｔａｃ启动子的启动下,在大肠杆菌中表达,ＲｕｂｉｓＣＯ基因片段在ｐＵＣ１９和ｐＫＫ２２３－３载体上的表达活性较高。进一步对ＲｕｂｉｓＣＯ基因表达产物进行了非变性聚丙烯酰胺凝胶电泳,检测到了ＲｕｂｉｓＣＯ蛋白质带。     

粟PsbA基因5’未端非编码区的结构特征

本文对粟（Ｓｅｔｃｒｉａｉｔａｉｌｉｃａ，俗称：谷子）叶绿体基因ｐｓｂＡ上２２ｋｂ的ＥｃｉＲＩ片段进行了克隆。该基因５’－未端非编码区就位于这个片段上。序列分析显示这个编码区存在着与原核生物基因类似的启动子结构：其“－１０”区序列为ＴＡＴＡＣＴ，与原核生物的仅相差一个核苷酸；“－３５”区序列为ＴＴＧＡＣＡ，与原核生物的完全相同。另一方面，在“－１０”和“－３５”区之间还存在着一个类似真核生物核基因启动子结构的“ＴＡＴＡＴＡ”保守序列。这表明粟ｐｓｂＡ基因的启动子既具有原核基因的特征、又具有真核基因的特征。粟ｐｓｂＡ基因的ｍＲＮＡ前导序多列长８７ｂｐ，与高粱的完全一致。可以推测：禾本科Ｃ３和Ｃ４植物中，ｐｓｂＡ基因ｍＲＮＡ前导序列区的差异可能具有某种普遍性。计算机分析结果显示，６种植物的ｐｓｂＡ基因ｍＲＮＡ前导序列区内均能形成小的茎环结构，而且这段“ＣＴＡＴＴＴＴ”额外序列恰好位于茎环结构中，造成了６种植物间茎环大小的差异。可能，这个小的二级结构对ｐｓｂＡ基因的表达调控有一定的影响。     

分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列,将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ３１酶切,Ｔ４ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒,将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶,酶活测定结果表明,基因表达水平比用原有的启动子高１－２倍,蛋白质分泌率在８４－９６％之间。     

人IL-18 cDNA克隆及其真核表达质粒的构建

用ＲＴ－ＰＣＲ技术从健康人外周血单核细胞的总ＲＮＡ中扩增出编码白细胞介素１８的全长ｃＤＮＡ,并将此基因定向克隆入真核表达质粒载体ｐｃＤＮＡ３中,通过对转化子的筛选得到了带有ＩＬ－１８插入片段的阳性克隆,经酶切分析及核苷酸测序表明,克隆到的基因与文献报道的完全一致。把重组质粒ｐｃＤＮＡ３／ＩＬ－１８分别转染人肝癌细胞ＨｅｐＧ２和鼠肉瘤细胞Ｓ１８０,在ｍＲＮＡ水平检测到ＩＬ－１８的表达,但ＩＬ－１８ 的表达未诱导肿瘤细胞产生ＩＦＮ－γ。     

Mn-SOD与抗癌胚抗原单链抗体基因的融合及高效表达

将Ｍｎ－ＳＯＤ与抗癌胚抗原（ＣＥＡ）单链抗体基因（Ｓｃ－Ｆｖ ｇｅｎｅ）融合,重组到含Ｔ７启动子的表达载体ｐＥＴ－２２ｂ（＋）中,构建表达质粒ｐＥＴＭｎ－ＳＯＤ－ＳｃＦｖ,并转化大肠杆菌ＢＬ２１（ＤＥ３）,进行高效表达,表达物占菌体可溶性总蛋白的２４％。ＳＤＳ－ＰＡＧＥ和蛋白质和迹图谱显示表达物分子量为４５ｋＤ与融合基因编码蛋白质的理论值相符。该蛋白质在大肠杆菌中为泌型表达有利于纯化。ＲＩＡ测定表     

Bt叶绿体转基因植株的抗虫性及后代表型分析

将Ｂｔ ＣｒｙＩＡ（ｃ） 基因与水稻（ Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．） 叶绿体ｐｓｂＡ 基因的启动子和终止子构建成表达盒,连同烟草（ Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ Ｌ．） 叶绿体基因组同源片段ｒｐｌ２＿ｔｒｎＨ＿ｐｓｂＡ和ｔｒｎＫ＿ＯＲＦ５０９Ａ 以及选择标记基因ａａｄＡ一起构建成烟草叶绿体转化载体ｐＴＲＳ８。基因枪法转化烟草叶片,经壮观霉素筛选获得转化再生植株。有些转基因植株对３龄棉铃虫（ Ｈｅｌｉｃｏｖｅｒｐａ ｚｅａ） 具有较强的毒杀作用,并能显著抑制昆虫蜕皮和生长发育。对高抗虫性植株的子一代（Ｔ１） 和子二代（Ｔ２） 进行的遗传学和分子生物学分析表明,Ｂｔ 基因已稳定地遗传给子代叶绿体,且抗生素抗性遗传遵循非孟德尔的母系遗传规律     

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.