培育具有安全选择标记或无选择标记的转基因植物

转基因植物中选择标记的安全性已成为植物基因工程研究的热点之一。从两个方面可以解决转基因植物中的选择标记问题。一是选用安全的正向选择标记,主要是与糖代谢和激素代谢相关的基因。二是构建能去除选择标记基因的转化系统,主要有共转化系统、双T-DNA边界载体系统、位点特异性重组系统和转座子系统等。这些植物基因工程的方法将有助于培育安全的转基因植物。 Abstract:The bio-safety of selective markers in transgenic plants has been a hot spot in the field of plant genetic engineering.To solve the problem of selective markers in the transgenic plants,two means of producing transgenic plants have been developed.One is the utilization of bio-safe positive selective markers which are genes mainly related to metabolism of auxins and carbohydrates.The other is the establishment of transformation systems allowing marker genes to be eliminated from the transgenic plants,which include co-ransformation,double T-DNA border vectors,site-specific recombination and transposition.All these approaches of plant genetic engineering will benefit breeding transgenic plants with bio-safety.     

用于叶绿体遗传转化的表达载体

叶绿体遗传转化是植物基因工程的新方向。本文简要介绍用于叶绿体遗传转化的表达载体的构建方法,涉及同源重组片段、叶绿体特异的启动子和终止子、筛选标记基因,以及目前在叶绿体中已实现表达的外源基因等内容。 Abstract:Chloroplast genetic transformation is a new way of plant genetic engineering.This paper reviews the construction methods of expression vector used in chloroplast genetic transformation.It contains the homologous recombinant fragments,the chloroplast specific promoter and terminater,the selectable marker genes and the interest genes whose expression in chloroplast have been achieved.     

水稻抗白叶枯病基因Xa-4的PCR标记研究

根据与水稻抗白叶枯病基因Xa-4紧密连锁的分子标记M55的序列设计引物,通过对国际水稻研究所育成的抗白叶枯病近等基因系和基因累加系的叶片DNA、半粒种子提取物及Xa-4基因的杂合体DNA的PCR特异扩增,初步建立了Xa-4的PCR标记体系。进而用该标记体系对我国籼型杂交水稻常用的亲本材料进行分析,揭示出了Xa-4在这些材料中的分布情况。 Abstract　Based on the sequence of a DNA marker tightly linked to the rice bacterial blight（BB） resistance gene Xa-4, two primers were designated and synthesized to develop a PCR marker for the gene. Specific amplified polymorphism analysis was carried out with these primers on a set of BB resistance isogenic lines and pyramided lines developed by IRRI. Two PCR bands were revealed corresponding to lines with dominant Xa-4 and those with the recessive allele, respectively, regardless the lines pyramided with other resistance genes. A hybrid with heterozygous Xa-4 produced both of the two allele PCR pattern. Then, the PCR marker was used to survey a range of hybrid rice germplasm. The results of the germplasm survey will be useful in hybrid rice breeding programs aimed at exploiting Xa-4.     

水产动物遗传连锁图谱的研究现状及应用展望

综述了近年来遗传连锁图谱在水产生物中的研究现状,包括作图群体、作图方法等,并对连锁图谱的应用前景作了展望,指出其在分子标记辅助育种、基因定位与克隆及比较基因组学等方面的应用潜力。 Abstract:Constructing genetic linkage map is an essential tool to acknowledge genome in aquaculture species.This paper has reviewed the current status of genetic linkage map research,including mapping population,mapping method and molecular markers used to construct linkage map.Linkage map has great potential in marker assisted selection (MAS),gene locating and cloning,and comparative genome mapping.Genetic linkage map with high density and wide coverage of genome will allow cloning the genes which contribute to economically important traits.The ultimate aim of the constructing linkage map is the development of fast-growing,disease-resistant strains of the major aquaculture species.     

降低转基因植物外源基因扩散的分子策略

转基因植物可以通过花粉或种子将外源基因转移到其他植物,从而对生态环境造成潜在的危害。如何降低外源基因的扩散已引起了人们的极大关注。目前降低外源基因扩散的方法主要有叶绿体转化、花粉不育、种子不育、闭花受精、无融合生殖、暂时性控制及转基因缓和等。主要对各种方法的原理和优缺点及目前的使用情况进行综述。Abstract: Transgenic plants can transfer foreign genes through pollen or seed to related plant species. This may cause potential harm to ecological environment. How to decrease the gene flow is drawing a growing public attention. The approaches for decreasing the gene flow include chloroplast transformation, pollen sterility, seed sterility, cleistogamy, apomixis, temporal control, and transgenic mitigation. The theoretical basis, advantages and disadvantages, and usage status of these approaches are presented in this review.     

丙肝病毒融合抗原基因NS3-C定点整合入衣藻 叶绿体基因组的研究

用PCR方法从丙型肝炎病毒(HCV) cDNA文库中克隆了两段DNA片段,即HC基因组非结构NS3区抗原基因(约0.7 kb)和核心抗原C区抗原基因(约0.6 kb)的cDNA片段。在两段cDNA间加入连接肽Ser-Pro-Gly-Ser的密码子序列,构建成融合抗原基因NS3-C。将该融合基因与衣藻叶绿体基因atpA的启动子和rbcL基因的3'末端连接,得到丙肝病毒融合抗原基因NS3-C表达盒,再将该表达盒与选择标记基因aadA表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体pSS6。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的PCR和Southern杂交分析表明,融合抗原基因NS3-C已整合到衣藻叶绿体基因组中。 Abstract:　Two DNA fragments encoding the nucleocapsid (C) region protein and the non-structural region 3 (NS3) protein of hepatitis C virus(HCV) were amplified from cDNA library by using PCR method. The 5' terminal of C cDNA fragment was linked up with the 3' terminal of NS3 cDNA fragment by a oligonucleotide linker Ser-Pro-Gly-Ser to form a chimeric gene NS3-C, which was placed under the control of the chloroplast atpA promoter and rbcL 3' region of Chlamydomonas reinhardtii to construct the chimeric gene NS3-C cassette. Then the NS3-C cassette was linked with selectable gene aadA cassette and the chloroplast homologous fragments of Chlamydomonas reinhardtii to generate transformation vector pSS6. Chloroplasts of Chlamydomonas reinhardtii were transformed by particle bombardment. Plastid transformants were selected by their resistance to 100 mg/L of spectinomycin. PCR and Southern hybridization analysis showed that the chimeric gene NS3-C had been integrated into chloroplast genome of Chlamydomonas reinhardtii.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

GFP基因在棉花转化中的应用

以绿色荧光蛋白GFP基因为报道基因,用花粉管通道和农杆菌介导的转化方法将外源基因导入棉花(Gossypium hirsutumL.),分别获得转化幼胚、幼苗和转化愈伤组织。用手持紫外灯结合显微镜检术能够快速地对转化子进行活体筛选鉴定,比用GUS检测方法有明显的优越性。本研究不但为花粉管通道转化法的可行性提供了新的证据,同时也建立了GFP用于棉花基因工程研究的检测技术体系。 Abstract：With the Green Fluorescent Protein gene (GFP) as a reporter gene, the transgenic embryos, seedlings and calli of cotton(Gossypium hirsutum L.) were obtained by the method of pollen tube pathway and Agrobacterium-mediated techniques separately. The GFP gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright?green fluorescence easily detectable and screenable in cotton tissue by fluorescence microscopy and a hand-held ultraviolet lamp. The screenable marker aided and facilated the rapid segregation of individual transformation events, drastically reduced the quantity of tissue to be handled. The GFP can be screened in vivo without destroying the materials, so it is more practical and useful than GUS. The use of GFP could advance the development of cotton gene engineering.     

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。     

去除转基因植物标记基因的研究进展

得到转基因植物以后 ,标记基因就失去了筛选的作用。但它的存在引起公众对转基因植物的安全性以及环境效应的担心 ,所以在目的基因转入后 ,要去除标记基因。该文主要就利用共转化、转座子、同源重组、位点特异重组酶等去除标记基因的方法进行了总结 ,并对各种方法的优缺点进行了比较 ,对该技术未来的发展趋势也进行了展望。     

以新霉素抗性基因突变体为筛选标志的真核表达载体的构建

新霉素抗性基因(neo)是真核表达载体的常用筛选标志neo基因编码新霉素磷酸转移酶Ⅱ（NPT Ⅱ）,能催化G418、卡那霉素等多种氨基糖苷抗生素分子磷酸化而使之失去抗菌活性。通过对真核表达载体的筛选标志基因neo进行定点突变,以降低NPTⅡ的活性,然后用含neo突变体的真核表达载体pmDNA构建荧光素酶表达质粒,稳定转染CHO-K1细胞,发现表达荧光素酶的阳性细胞比例达到95%,其中高表达细胞集落的筛选率明显高于对照组。     

转基因植物中标记基因研究概况

标记基因在植物基因工程中具有重要的作用。它在遗传转化中的关键作用是区分转化和非转化的细胞,以筛选并鉴定出转化的细胞、组织和转基因植株。目前,已报道的标记基因种类很多,划分标准也各不相同。出于对生态环境和转基因食品的生物安全性考虑,从传统的选择标记基因、与激素代谢相关的基因、与氨基酸代谢相关的基因、与糖类代谢相关的基因、能解除化合物毒性（或胁迫）的基因、编码能产生特定荧光物质的蛋白酶类的基因、利用颜色差异性筛选转化体的相关基因及抗性标记基因的敲除技术八个不同的方面,综述了标记基因的种类、作用原理、应用价值及存在的问题。在标记基因的综合应用方面,详细总结了标记基因与组织（或器官）特异性启动子和MAT载体系统的结合应用,以及P．葡萄糖苷酸酶作为多功能标记基因的综合应用。最后,对标记基因的发展前景进行了探讨分析。     

Use of the TRP1 auxotrophic marker for gene disruption and phenotypic analysis in yeast: a note of warning

The TRP1 marker has been commonly used for gene disruption experiments and subsequent phenotypic analysis. However, introduction of the TRP1 gene into a trp1 strain markedly affects growth under many conditions used for phenotypic profiling. Therefore, its use in the past should be revisited and utilization of this marker should be avoided in future analyses.     

乔丹基因:在团藻中发现的跳跃基因

Ｄａｖｉｄ Ｋｉｒｋ 和 Ｓｔｅｐｈｅｎ Ｍ ｉｌｌｅｒ 最近用一种特殊的转座子 乔丹基因作为标记克隆了两个控制团藻发育的重要基因：ｇｌｓ Ａ 基因和 ｒｅｇ Ａ 基因⒚他们的这一工作可能为遗传学研究开辟了一条新的道路⒚     

hpt与bar基因作为水稻转基因筛选标记的比较研究

标记基因的选择是影响植物遗传转化和转基因后代筛选成败的关键因素。hpt与bar作为两种常用的水稻转化筛选标记被广泛应用于水稻的转化。为比较两者在实际应用中的效果, 文章首先对比了在潮霉素和除草剂(Bialaphos)两种筛选剂下水稻遗传转化的情况。研究表明, hpt基因的转化筛选体系相对于bar基因在转化效率上提高近两倍, 转化周期提前至少10 d, 且插入基因拷贝数更低。随后, 文章分析了利用潮霉素浸种法在田间筛选转基因水稻的可行性, 研究显示当潮霉素浓度大于167 mg/L时, 可以对以水稻品种kitaake为亲本的转基因材料进行有效筛选, 达到常规除草剂的筛选效果。但与除草剂相比较, 潮霉素的田间筛选成本却处于劣势。文章研究和讨论了hpt和bar基因在遗传转化和后代田间筛选中的优缺点, 并提供了一种利用潮霉素浸种法筛选转基因后代阳性植株的手段, 为将来在水稻转基因研究工作中根据实际需求选择合适的遗传转化、筛选体系提供参考。     

酿酒酵母SNF4基因敲除缺失菌株的构建

构建一株酿酒酵母SNF4基因缺失菌株并研究其对乙醇产量的影响。扩增带有SNF4基因上下游同源序列和Kanr筛选标记的SNF4基因敲除组件,转化到酿酒酵母YS2获得阳性克隆子,然后将质粒pSH65转到阳性克隆子中,半乳糖诱导pSH65表达Cre酶切除Kanr筛选标记,获得SNF4等位基因完全缺失菌株YS2-△SNF4。发酵实验结果表明,缺失菌株YS2-△SNF4乙醇产量较出发菌株提高了7.57％。利用Cre-LoxP系统,成功构建了SNF4等位基因完全缺失菌株并提高乙醇产生量。     

小麦抗白粉病基因Pm23的RAPD标记

小麦抗白粉病基因Pm23对世界上很多麦区流行的白粉病表现高抗或免疫.本研究以Pm23和Chancellor为抗感亲本,用集群分离分析法对抗性基因Pm23进行了RAPD分析,从320个十碱基随机引物中筛选到一个与Pm23紧密连锁的相引相标记OPE051100. 对F2分离群体进行RAPD分析表明,该标记与Pm23基因之间的连锁距离为10.65±3.25 cM.该标记可以有效用于小麦育种分子标记辅助选择中.