可删除非目的基因表达载体的构建

[目的]通过构建能够删除选择标记基因的表达载体,消除由非目的基因带来的影响,更好地研究转入基因的单一功能。[方法]利用Cre/LoxP位点特异性重组系统,对DsRed2-1载体进行改造,分别引入LoxP序列及多克隆位点序列,并引入TK基因进行负筛选,最终获得无目的基因的表达载体。[结果]诱导的载体片段在分子水平上发生了重组,载体转染细胞在细胞水平上也产生了特异性红色荧光。[结论]Cre/loxP系统的标记基因诱导删除体系切实可行,有着广泛的应用前景。     

基于Cre/loxP重组系统的多基因载体构建及烟草转化研究

利用一套基于Cre/loxP重组系统的植物多基因表达载体系统将苏云杆菌毒蛋白基因(BtCryIAc)、甜菜碱醛脱氢酶基因(BADH)、GA20氧化酶基因(pttGA20ox)和rolB基因构建于植物表达载体pYL1305上,命名为pYL1305BBGR。采用农杆菌介导的叶盘法转化烟草,经PCR和Southern blot检测发现,多基因已成功转入烟草基因组中;经半定量RT-PCR检测证实,4个基因均能正常表达。     

Cre/loxP位点特异性重组系统在植物中应用的研究进展

Cre/loxP位点特异性重组系统自发现以来,在植物基因重组领域得到了广泛应用,已成为提高转基因植物安全性的有效方法。本文介绍了该系统的基本概况及其在转基因植物表达载体构建方面的应用,尤其是在基因删除、定点整合、多基因表达以及杂种优势利用等方面的应用做了重点阐述。     

Cre／loxp重组系统在转基因植物中删除标记基因的应用

在植物遗传转化中,标记基因是必须的,但标记基因的存在引起人们对转基因食品安全性的关注。Cre／loxp重组系统可用于删除转基因植物中的标记基因,可以通过杂交或二次转化,或瞬时表达、诱导表达、组织特异表达重组酶来获得无标记基因的转基因植株。     

利用Cre/lox定位重组系统获得无选择标记GFP转基因烟草

【目的】利用Cre/lox系统具有的特异重组特性,以绿色荧光蛋白GFP为目的基因,获得无选择标记的GFP转基因烟草。【方法】将Bar基因表达盒置于两个同向lox位点之间并与GFP基因表达盒融合后获得相应植物表达载体,转化烟草后获得抗除草剂Basta的GFP转基因植株。GFP转基因植株叶盘二次转化导入重组酶Cre基因后,实现与GFP基因连锁的Bar基因表达盒剔除,剔除Bar基因的植株开花后自交使重组酶Cre基因分离,获得无选择标记的GFP转基因烟草。【结果】将含Bar基因表达盒以及GFP基因表达盒的植物表达载体转化烟草Wisconsin 38后获得了抗除草剂Basta以及GFP荧光表达的转基因植株。随机抽取5株转基因植株二次转化导入Cre基因,所获得的再生植株叶盘进行Basta的抗性检测,绝大多数单株对应叶盘在含8 mg•L-1 PPT（phosphinothricin）的筛选培养基上无法再生死亡,删除效率在76%～100%。对Bar基因删除后区域片段进行克隆测序分析显示,Bar基因表达盒已经被精确删除。Bar基因删除植株开花后自交,获得的自交后代进行NPTⅡ抗性检测,NPTⅡ敏感植株分子检测显示均只含有GFP基因。【结论】利用Cre/lox系统获得烟草无选择标记的转基因植物是稳定可行的,可广泛应用于其它无选择标记转基因植物的培育。     

利用Cre/lox重组系统建立番茄基因工程雄性不育恢复系

 【目的】利用Cre/lox重组系统具有的重组删除特性建立番茄工程恢复系,特异删除F1中的雄性不育基因恢复其育性。【方法】将TA29-Barnase雄性不育基因表达盒置于两个同向lox位点之间并与NPTⅡ基因、Bar基因融合后获得植物表达载体pBinBarloxTABn,转化番茄获得雄性不育转基因植株。Cre基因在 CaMV35S启动子的驱动下转入番茄获得工程恢复系。两者在开花时进行杂交,利用Cre重组酶删除F1中的TA29-Barnase不育基因表达盒使育性恢复。【结果】利用NPTⅡ作为转化筛选标记基因获得了番茄雄性不育转基因植株。转基因植株中的Bar基因能正常表达,真叶叶盘在含PPT 3 mg?L-1（phosphinothricin）分化培养基上能分化愈伤组织及芽;真叶具有抗PPT 20 mg?L-1浓度以上的能力。获得的TA29-Barnase转基因植株表现雄蕊退化、无花粉产生或产生少量形状畸形且无生活力的花粉。雄性不育植株自花授粉不能坐果,用非转基因保持系花粉授粉后,果实正常膨大结籽,杂交后代对除草剂Basta的抗性按1﹕1分离。不育植株与Cre转基因工程恢复系杂交后,果实也正常膨大结籽。对不育植株与Cre转基因工程恢复系杂交后代进行分子检测,结果发现同时含Bar 基因和Cre基因F1植株中的TA29-Barnase雄性不育基因被精确删除。TA29-Barnase基因删除植株育性被恢复,能正常开花结果。【结论】利用Cre/lox重组系统建立的Cre工程恢复系成功将番茄F1代中的不育基因删除,恢复了 F1的育性。该研究结果为植物基因工程雄性不育系的育性恢复提供了一条新的途径。     

Cre／loxp位点特异性重组系统在转基因植物中的应用

位点特异性重组系统能够在植物遗传转化中更准确、可靠的操纵外源DNA的引入或删除,已成为植物遗传操作中的重要工具。本文简要介绍目前应用最为广泛的Cre/loxp位点特异性重组系统的重组机制,并着重阐述Cre/loxp系统在删除转基因植物中标记基因及定点整合等方面的应用。     

Cre/LoxP系统介导的位点特异性重组技术

Cre/LoxP系统是一种对转入基因进行定点操作的基因重组系统,在遗传操作中具有重要作用。该系统可以将外源基因定点整合到染色体上或将特定DNA片断删除。     

转基因植物中删除选择标记基因的研究进展

选择标记基因广泛应用于植物转基因工程中,然而这些基于除草剂和抗生素类抗性选择标记基因的利用,在转基因筛选和鉴定完成后功能多余。当前转基因植物的标记基因及相关产物对环境污染和食用安全的潜在影响,已经引起了人们的广泛关注。随着转基因技术的不断发展和人们对转基因植物和食品安全性的关注,删除转基因植物中的选择标记基因非常重要。文章着重讨论了植物转基因过程中,选择标记基因的利用,删除选择标记基因的几种方法,包括共转化法、位点特异性重组、染色体内重组、转座子介导的重组、删除叶绿体转化中选择标记基因,以及无选择标记基因植株的直接再生等。同时,还对这些技术的发展作了展望。     

酿酒酵母 ERG6基因缺失突变株的构建

设计含有与ERG6基因两侧序列同源的长引物,以质粒pBlue-Kan为模板进行PCR扩增,构建含有Cre/lox P系统的酿酒酵母ERG6基因敲除组件.将基因敲除组件转化至酿酒酵母(Saccharomyce cerevisiae)AD1-8,通过同源重组的方式使目的基因缺失,获得为lox P-Kan-lox P序列组件所替换而产生Kanr的阳性克隆子.然后再将质粒pHis-Cre转入阳性克隆子表达Cre重组酶敲除筛选标记,成功获得酿酒酵母ERG6基因缺失的突变株,并命名为AD1-8-δ.     

Cre/Loxp重组酶系统与转基因

Cre/Loxp重组酶系统是一种对转入基因进行组织特异性、定点操作的基因重组系统。该系统可以迅速有效地进行基因的整合性删除、条件性重组、染色体易位等方面的研究,从而可以将其应用于转基因动物和植物领域。     

Construction of Marker-Free GFP Transgenic Tobacco by Cre/Iox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.     

Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper (Capsicum annuum L.). Chili pepper inbred lines (A, D, E, and I) were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR [MB (MS mineral+vitamine B5)+BA (6-Benzyladenine) 5.0 mg L-1 +IAA (indoleacetic acid) 1.0 mg L-1 +GA3 (gibberellic acid) 1.0 mg L-1 + sucrose 3% +agar 6.5 g L-1] for 2 d. The explants were infected by Agrobacterium tumefaciens when their OD600 (optical density at 600 nm) reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB + BA 5.0 mg L-1 + IAA 1.0 mg L-1 + GA3 1.0 mg L-1 + sucrose 3% + agar 6.5 g L-1 + AS (acetosyringone)200 μmol L-1], these cotyledons with petiole were cultured on selective differentiation medium in the media MT [MB medium supplemented with BA [5.0 mg L-1 + IAA 1.0 mg L-1 + GA3 1.0 mg L-1 + AgNO3 5.0 mg L-1 + CW (coconut water) 5% +Km (kanamycin) 65 mg L-1 + Cb (carbenicillin) 500 mg L-1 + 3% sucrose + agar 6.5 g L-1]. The Kmr (kanamycin resistant) bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.     

Study on Agrobacterium-Mediated Transformation of Pepper with Barnase and Cre Gene

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper （Capsicum annuum L.）. Chili pepper inbred lines （A, D, E, and I） were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR[MB（MS mineral＋vitamine B5）＋BA（6-Benzyladenine） 5.0 mg·L^-1 ＋IAA（indoleacetic acid） 1.0 mg·L^-1＋GA3（gibberellic acid） 1.0mg·L^-1＋sucrose 3%＋agar 6.5g·L^-1] for 2d. The explants were infected by Agrobacterium tumefaciens when their OD600（optical density at 600 nm）reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB＋BA5.0 mg·L^-1＋IAA 1.0 mg·L^-1 ＋GA3 1.0 mg·L^-1＋sucrose 3% ＋agar 6.5 g·L^-1＋AS （acetosyringone） 200μmol·L^-1, these cotyledons with petiole were cultured on selective differentiation medium in the media MT[MB medium supplemented with BA [5.0 mg·L^-1＋ IAA 1.0 mg·L^-1＋ GA3 1.0 mg·L^-1＋ AgNO3 5.0 mg·L^-1＋ CW （coconut water） 5% ＋ Km （kanamycin） 65 mg·L^-1＋ Cb （carbenicillin） 500 mg·L^-1＋ 3% sucrose ＋ agar 6.5 g·L^-1].The Kmr （kanamycin resistant） bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar results were obtained from in vitro pollen germination experiments.