通过构建三段T-DNA载体高频率获得无标记转基因大豆植株的研究

高频率获得无选择标记转基因植株有利于转基因植物的环境释放和安全性生产,农杆菌介导的共转化法是获得无标记转基因植株的方法之一。含二段T-DNA载体的共转化法已被人们成功应用,而二段以上T-DNA载体的共转化法还未见报道。基于这一目的,通过几个中间质粒构建了含有三段T-DNA的双元表达载体pNB35SVIP1,其中包含1个拷贝bar基因选择标记基因表达盒和2个拷贝VIP1目的基因表达盒。利用EHA101农杆菌菌系介导法转化大豆子叶节,经过在含3～5mg/L glufosinate培养基上多次筛选,获得了一定数量抗性再生植株,然后对抗性再生植株进行叶片涂抹除草剂、Southern blot和Northern blot检测,共鉴定出51棵T₀代转基因植株,转化频率0.83%～3.16%,二个基因的共转化频率为86.4%。在对T₁代群体进行叶片涂抹除草剂检测的基础上,不抗除草剂植株进行PCR、Southern blot和Northern blot检测,共鉴定出41棵无选择标记转基因植株,无标记植株获得率为7.6%。检测结果还表明,T₁代群体中22.7%的株系发生了基因丢失现象,27.3%的株系发生了bar基因沉默现象,目的基因在37.1%的无标记植株中发生了沉默现象。三段T-DNA的双元表达载体是获得无标记转基因植株的理想途径     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法.通过体外重组构建了双T-DNA双元载体pDLBRBbarm.载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA.利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%.对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ.这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物.研究还利用位于2个不同载体上的nptⅡ基因与 bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%～47.4%,低于使用双T-DNA转化的共转化频率.     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法。通过体外重组构建了双T-DNA双元载体pDLBRBbarm。载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA。利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59．2％。对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19．5％的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ。这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物。研究还利用位于2个不同载体上的nptⅡ基因与bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20．0％～47．4％,低于使用双T-DNA转化的共转化频率。     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

表达γ-亚麻酸的无筛选标记转基因大豆的鉴定

对所获得的9株无筛选标记基因的T1代莆豆8008转基因大豆的T2代进行了分析。PCR和Southern杂交检测表明标记基因已不存在于转基因株系中,同时目的基因以纯合或者杂合状态存在于部分株系中,除草剂抗性分析也表明这些转基因株系已不再具有抗除草剂的能力。Northern杂交检测表明Δ6-脂肪酸脱氢酶基因在转基因株系中可以转录。GC分析表明其中6个转基因植株富含GLA。     

利用Ac/Ds转座子系统在水稻中获得无选择标记转基因植株的方法

获得无选择标记转基因植株是进行重复转基因及消除转基因植株中标记基因潜在危害性的关键。实验采用了Ac／Ds转座子系统在水稻(Oryza sativa,L．)中进行无hpt选择标记的转基因。将含有目的基因bar的Ds元件和hpt标记基因置于同一个T-DNA中,通过农杆菌(Agrobacterium tumefaciens)EHAl05介导将Ac-T-DNA及Ds-T-DNA分别转入到不同的水稻植株,再将单拷贝的Ac-T-DNA植株与单拷贝的Ds-T-DNA植株杂交得到同时含有Ac和Ds元件的F1植株,Fl自交产生F2后代,F2植株中转座后的Ds元件与T-DNA独立分离,在总共100株F2水稻植株中筛选得到2株只含有Ds元件插入而无hpt标记基因的转基因水稻植株。结果表明,利用Ac／Ds转座子系统在水稻中获得无选择标记的转基因植株是可行的。     

导入bar基因的水稻新植株可遗传

江苏省农业科学院粮作所王才林、赵凌、宗寿余等和中科院上海生化所龚蓁蓁等9位科研人员,用花粉管通道法将bar基因导入水稻获得可遗传的转基因植株。亦即他们利用花粉管通道法将抗Basta除草剂的bar基因导入水稻品系E32,获得转基因植株。但在T0代仅表现为部分抗性,T1代全部获得了抗性,T3代全部转基因植株能充分表达对Basta除草剂的抗性。通过对转基因植株后代进行PCR分析,证实了bar基因已整合到受体植株的基因组之中,以后又经过遗传分析表明,bar基因能在有性生殖过程中传递给后代植株,并在T3代开始就可分离出抗性一致的稳定株系。目前,其…     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

转bar基因小麦和非转基因小麦抗除草剂鉴定方法比较

方便、快捷、准确地对转基因小麦中的bar基因进行检测,对于筛选纯合稳定转基因植株、获得无筛选标记转基因植株、鉴定常规小麦品种和商品小麦中的bar基因成分等具有一定价值。本试验对叶片涂抹、植株喷洒、培养基添加除草剂3种方法鉴定转bar基因小麦植株的效果进行了比较,表明3种方法都能很好鉴定转基因小麦中的bar基因,叶片涂抹200mg/LLiberty鉴别的准确性高于PCR检测,植株喷洒Basta的适宜浓度为100mg/L,喷洒Liberty的适宜浓度为150mg/L,培养基添加Bialaphos的适宜浓度为5～8mg/L。叶片涂抹和植株喷洒除草剂方法受环境条件影响较大,区别转基因植株和非转基因植株的标准不够明确。相比之下,成熟胚离体培养除草剂筛选不受外界环境条件的影响,具有鉴定效果直观明了、操作简单、试验周期短等优点,在检测小麦转入或飘入的bar基因方面具有潜在应用前景。     

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.