昆虫抗冻蛋白基因的克隆与表达研究进展

产生抗冻蛋白(antifreeze protein,AFP)是许多昆虫抵御寒冷的一种重要机制。昆虫抗冻蛋白基因的克隆和表达是研究抗冻蛋白活性和功能的主要途径。文章归纳GenBank所登录的昆虫抗冻蛋白基因及其特点,总结昆虫抗冻蛋白基因的天然表达和基因工程表达方面尚未明确或需要克服的一些问题。目前在GenBank注册的昆虫抗冻蛋白基因约100个,集中于9种昆虫隶属鞘翅目3个科和鳞翅目1个科。昆虫抗冻蛋白基因具有多拷贝和多同种型(isoforms)的特点。昆虫抗冻蛋白的天然表达具有物种间和同种型间的多样性。基因工程表达昆虫抗冻蛋白需要克服表达量低活性不高的问题。对昆虫抗冻蛋白表达规律的研究有助于全面认识其功能。     

抗冻蛋白结构基因片段的克隆

为了研究和开发利用抗冻蛋白或多肽,木文通过聚合酶链式反应（ＰＣＲ）合成了抗冻蛋白１１０ｂｐ的结构基因片段,然后将该基因片段克隆到大肠杆菌质粒载体Ｐ（Ｂｌｕｅｓｃｒｉｐｔ）Ⅱｋｓ＋／－上,获得了重组质粒,经酶切证明,获得的重组质粒中含有抗冻蛋白结构基因片段,以供该基因在大肠杆菌或酵母中表达抗冻蛋白。     

用鱼抗冻蛋白生产抗霜冻植株

加拿大国立研究协会植物生物技术研究所的F.Georges等人开发了一种化学合成编码冬比目鱼抗冻蛋白基因的新方法.借助于电激法把合成基因引入玉米原生质体,并在此原生质体中得到表达,在无细胞提取液中明显地鉴定到该抗冻蛋白. 抗冻蛋白基因合成的第一步是:通过把抗冻蛋白基因引入冷冻敏感性植株里从而使植株产生抗霜冻性.作者认为,此合成方法可广泛地应用于化学合成基因.     

抗冻蛋白研究进展

抗冻蛋白是一类具有热滞效应、冰晶形态效应和重结晶抑制效应的蛋白质。简单介绍了各种抗冻蛋白的生化特征、作用机制及其应用研究 ,并对抗冻蛋白的基因和基因工程研究作了较为系统的综述。     

荒漠甲虫光滑鳖甲和小胸鳖甲抗冻蛋白基因及其3'-UTR的序列分析

拟步甲科昆虫光滑鳖甲Anatolica polita Kaszab和小胸鳖甲Microdera punctipennis Kaszab分别是新疆准噶尔盆地荒漠地区的广布种和特有种。它们的生活习性有很大差异,光滑鳖甲白天活动,且活动范围广,而小胸鳖甲喜阴避光夜晚活动,活动范围小,但在两者体内都发现有多拷贝的抗冻蛋白基因,并且抗冻蛋白基因在夏季也有表达。为了比较这两种昆虫的抗冻蛋白序列以及不同时期表达的抗冻蛋白序列的差异情况,本研究分别以光滑鳖甲和小胸鳖甲的过冬和夏季成虫为材料,根据已克隆的抗冻蛋白基因序列设计通用引物,扩增新的抗冻蛋白基因及其3'-非翻译区(3'-UTR)。序列分析结果表明,无论是冬季还是夏季表达的抗冻蛋白在两种昆虫中都具有拟步甲科昆虫抗冻蛋白的特点,即含有不同数量的"TCTXSXXCXXAX"12个氨基酸的重复序列;光滑鳖甲抗冻蛋白基因的变异性大于小胸鳖甲,并且在重复基序的保守位点都有突变现象。小胸鳖甲抗冻蛋白在C末端有较大变异。光滑鳖甲抗冻蛋白基因的3'-UTR区变异较大,而小胸鳖甲抗冻蛋白基因的3'-UTR区几乎没有变异。推测两种昆虫抗冻蛋白及抗冻蛋白基因3'-UTR的变异性可能与抗冻蛋白的表达调控以及昆虫对微环境的适应性有关。     

黄粉虫不同抗冻蛋白基因家族成员的低温应激表达

黄粉虫Tenebrio molitor L.抗冻蛋白基因家族有多个成员,其氨基酸数量和蛋白结构存在差异。尽管有报道发现冷驯化后这些抗冻蛋白的表达量会升高,但不同家族成员是否存在功能分化尚不清楚。本研究中,检测了冷驯化对低温死亡率的效应和对不同类型的抗冻蛋白家族成员基因表达量的影响。结果表明,冷驯化可以显著降低黄粉虫幼虫的低温死亡率和提高不同类型抗冻蛋白基因的表达量。其中,长的抗冻蛋白和低温死亡率的相关关系最为明显。说明不同的抗冻蛋白家族成员的功能有明显的分化,为进一步理解抗冻蛋白的活性和利用抗冻蛋白提供了新的认识。     

昆虫抗冻蛋白的研究

抗冻蛋白是具有热滞活性,能结合并抑制冰晶生长和抑制冰的重结晶的一类蛋白质。近几年来,昆虫抗冻蛋白的研究取得了较快的发展,本文通过分析昆虫抗冻蛋白的结构特点、抗冻活性、作用机制,并讨论了抗冻蛋白在食品工业、医学、基因工程方面的应用。结果表明,昆虫抗冻蛋白虽然结构呈多样性,但有很多关键的残基具有保守性,对维持抗冻蛋白结构和功能的完整性发挥着重要的作用;抗冻蛋白是由多基因家簇编码的。其作用机制主要是吸附一抑制机制,抗冻蛋白依靠氢键吸附到冰晶格,抑制冰晶生长;昆虫抗冻蛋白的应用具有很广阔的前景。     

用美洲拟鲽抗冻蛋白基因转化植物的问题

评估了我们用美洲拟鲽抗冻蛋白基因转化烟草和国内有人用同一基因转化蕃茄的研究,提出植物耐寒性状的提高与转基因植物中抗冻蛋白含量呈正相关是转基因植物成功的关键性指标。同时,美洲拟鲽抗冻蛋白的空间结构与冷诱导蛋白相似而且都保护细胞膜免受冻害,因而提出了用冷诱导蛋白调节元的顺式作用元件和反式作用因子的调控序列来控制抗冻蛋白基因在植物中冷诱导表达的思路。     

Gene expression of different antifreeze proteins of Tenebrio molitor in response to cold acclimation

黄粉虫Tenebrio molitorL.抗冻蛋白基因家族有多个成员,其氨基酸数量和蛋白结构存在差异.尽管有报道发现冷驯化后这些抗冻蛋白的表达量会升高,但不同家族成员是否存在功能分化尚不清楚.本研究中,检测了冷驯化对低温死亡率的效应和对不同类型的抗冻蛋白家族成员基因表达量的影响.结果表明,冷驯化可以显著降低黄粉虫幼虫的低温死亡率和提高不同类型抗冻蛋白基因的表达量.其中,长的抗冻蛋白和低温死亡率的相关关系最为明显.说明不同的抗冻蛋白家族成员的功能有明显的分化,为进一步理解抗冻蛋白的活性和利用抗冻蛋白提供了新的认识.     

用美洲拟鲽抗冻蛋白基因转化植物的问题

评估了我们用美洲拟鲽抗冻蛋白基因转化烟草和国内有人用同一基因转化蕃茄的研究 ,提出植物耐寒性状的提高与转基因植物中抗冻蛋白含量呈正相关是转基因植物成功的关键性指标。同时 ,美洲拟鲽抗冻蛋白的空间结构与冷诱导蛋白相似而且都保护细胞膜免受冻害 ,因而提出了用冷诱导蛋白调节元的顺式作用元件和反式作用因子的调控序列来控制抗冻蛋白基因在植物中冷诱导表达的思路。     

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.