NaCl胁迫对转基因杨树外源基因表达的影响

以具高抗虫性的转抗虫基因‘741杨’及在此基础上转入了发根农杆菌Ri质粒T-DNA株系的组培苗为材料,研究了转基因株系BtCrylAc抗虫基因和发根基因的表达及其对NaCl胁迫的反应。结果表明,转入Ri质粒T- DNA上的rol基因后,导致苗木根系数目增加,根系长度减小,IAA和GA含量显著提高,抗虫BtCrylAc基因编码的毒蛋白的表达量降低;随着NaCl胁迫强度的增加,苗高、根系数量、叶绿素含量及IAA、GA含量逐步降低,而根系的长度加大,Bt毒蛋白含量显著提高,表明NaCl胁迫使转基因杨外源Bt毒蛋白基因的表达增强,而发根农杆菌Ri质粒T-DNA的表达下降。     

转抗虫基因三倍体毛白杨植株体内农杆菌残存与逃逸

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（APLA）基因构建的双抗虫基因表达载体,通过农杆菌介导法对三倍体毛白杨进行了转化,对转化后植株体内残存农杆菌在继代培养和移栽过程中进行了跟踪检测。结果表明：通过对转化再生植株的分子生物学检测,42个株系中,33个株系为阳性,阳性率达到80％;用Bt毒蛋白抗血清进行ELSA检测结果表明,7个转基因株系都有Bt杀虫蛋白表达;基因转化后,可采用附加50mg／L卡那霉素,300mg／L羧苄青霉素的筛选培养基消除细菌并进行抗性芽筛选。对28个转基因株系叶片、茎段和根段在含有卡那霉素50mg／LYEB培养基上进行细菌培养,通过在T-DNA区、质粒Vir区和农杆菌基因组设计引物,进行PCR检测,证明有3个株系（33、37、5号）检测到残存工程农杆菌,并在组培瓶中存活24个月。将带菌的3个株系组培苗移栽到花盆中,室内培养1个月后,在33号株系根际土壤中检测到了目的农杆菌。     

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体,通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.,获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Western Blot检测,同时用转基因株系叶片对杨扇舟蛾Clostera anachoreta Fabricius和舞毒蛾Lymantria dispar L.幼虫进行室内饲虫试验,并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中,对杨扇舟蛾高抗株系占总参试系号的41％,中抗系号占35.0％,低抗系号占24％,对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育,且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明,不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性,并存在极显著相关。ELISA检测结果表明,不同转基因株系Bt毒蛋白表达量存在差异,变化在0.0011％～0.0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关,表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性,但与抗虫程度相关不明显,单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。     

转抗虫基因三倍体毛白杨植株体内农杆菌残存与逃逸

用部分改造的 BtCry1Ac基因与慈菇蛋白酶抑制剂(API-A)基因构建的双抗虫基因表达载体,通过农杆菌介导法对三倍体毛白杨进行了转化,对转化后植株体内残存农杆菌在继代培养和移栽过程中进行了跟踪检测。结果表明：通过对转化再生植株的分子生物学检测,42个株系中,33个株系为阳性,阳性率达到80％;用Bt毒蛋白抗血清进行ELSA检测结果表明,7个转基因株系都有Bt杀虫蛋白表达;基因转化后,可采用附加50 mg/L卡那霉素,300 mg/L羧青霉素的筛选培养基消除细菌并进行抗性芽筛选。对28个转基因株系叶片、茎段和根段在含有卡那霉素50 mg/L YEB培养基上进行细菌培养,通过在T-DNA区、质粒Vir区和农杆菌基因组设计引物,进行PCR检测,证明有3个株系（33、37、5号）检测到残存工程农杆菌,并在组培瓶中存活24个月。将带菌的3个株系组培苗移栽到花盆中,室内培养1个月后,在33号株系根际土壤中检测到了目的农杆菌。     

转单、双Bt基因741杨外源基因表达和抗虫性比较

【目的】研究联合使用两种或两种以上的抗虫基因的抗虫效果, 同时鉴定并筛选出转双Bt基因741杨对鳞翅目和鞘翅目害虫有较强抗性的株系。【方法】选取转三基因（Cry3Aa+Cry1Ac+API）741杨8个株系、 转双基因（Cry1Ac+API）741杨1个株系和转单基因（Cry3Aa）741杨3个株系为试材, 从外源基因PCR检测、 毒蛋白表达和抗虫性三方面对转基因株系进行对比分析。【结果】经PCR扩增后各转基因株系出现了预期的电泳条带。ELISA蛋白检测显示转基因株系中都有与所含基因相应的Bt杀虫蛋白表达。用转基因株系新鲜叶片进行柳蓝叶甲Plagiodera versicolora和美国白蛾Hyphantria cunea室内饲虫实验表明： 转入不同抗虫基因的杨树对昆虫的抗性具有选择性, 对非靶标昆虫没有毒杀作用。转双Bt基因741杨具有双抗性, 不同转基因株系表现出高中低的抗性水平： 在对柳蓝叶甲的抗性上, 筛选出的其中5个高抗株系（pCCA1, pCCA2, pCCA5, pCCA6和pCCA9）的抗性水平明显比含Cry3Aa单Bt基因的3个高抗株系（pCC11, pCC53和pCC84）高; 在对美国白蛾的抗性上, 有7个株系（pCCA2~pCCA7和pCCA9）的抗性水平与含Cry1Ac单Bt基因株系（pB29）表现一致, 只有1个株系（pCCA1）对美国白蛾表现出了极低的抗性。【结论】多个抗虫基因在741杨上的联合使用, 不仅扩大了抗虫谱, 其中的高抗株系还具有了更高的抗虫能力, 有效地发挥了基因的叠加效应。     

转不同rol基因741杨株系相关性状比较分析

对已转入rolB、rolC基因741杨株系进行分子检测,并对其根系生长状况、苗高、叶绿素a和b、叶绿素荧光以及内源激素等指标进行测定。结果表明,转rolB、rolC基因741杨各株系发根率、发根量均高于未转基因对照。转rolB基因741杨株系苗高与对照无显著差异,叶绿素a、b含量上升,Fv/Fm、PI值下降,IAA、ZT含量上升,ABA含量下降。转rolC基因741杨各株系顶端优势减弱,植株矮化,叶绿素a、b含量降低,Fv/Fm、PI值下降,IAA、ZT、ABA含量上升。总体比较,转rolB基因741杨株系生根率,叶绿素a、b含量,PI值、ZT和IAA含量均高于转rolC基因741杨株系。     

苏云金杆菌毒蛋白抗虫基因工程取得重大进展

害虫是作物的天敌。长期以来,化学杀虫剂在防治虫害方面发挥了重要的作用。但是,化学杀虫剂对环境的污染和人类健康的危害越来越受到人们的关注,因此,科学家们更加致力于生物防治特别是利用基因工程进行抗虫杀虫的研究。随着植物基因工程技术的发展,将苏云金杆菌毒蛋白(Bt毒蛋白)基因转入植物获得抗虫的转基因植物已成为可能。1987年,国外有三家实验室报道了将Bt毒蛋白基因转入烟草或蕃茄,获得抗虫植株。但表达量低,抗虫活性不高。中国科学院微生物所等单位的研究人员承担了国家“七五”攻关课题——苏云金杆     

发根农杆菌转化龙胆再生植株的研究

本文利用具Ri质粒的发根农杆菌(Agrobacterium rhizogenes)通过叶盘法对药用植物龙胆(Gentiana manshurica Kitagawa)进行了转化实验,发根农杆菌15834感染龙胆,诱发产生毛状根,并得到再生的龙胆植株,冠瘿碱检测实验表明,再生植株显示甘露碱带(mannopine),说明发根农杆菌Ri质粒的T-DNA部分已转移到龙胆植物细胞中。再生的龙胆丛生苗可以在不含激素的简化培养基上快速无限繁殖,转化的龙胆植株明显具有发达的根系,且根部龙胆苦甙含量比对照高,从而为东北龙胆栽培事业的发展以及龙胆有效成份的工业化生产提供了新的途径。     

发根农杆菌转化龙胆再生植株的研究

本文利用具Ri质粒的发根农杆菌(Agrobacterium rhizogenes)通过叶盘法对药用植物龙胆（Genriana manshurics Kitagaua）进行了转化实验,发根农杆菌15834感染龙胆,诱发产生毛状根,并得到 再生的龙胆植株。冠瘦碱检测实验表明,再生植株显示甘露碱(mannopine)带,说明发根农杆菌Ri质 粒的T-DNA部分已整合到龙胆植物细胞中。再生的龙胆丛生苗可以在不含激素的简化培养基上快速 繁殖,转化的龙胆植株具有发达的根系,根部龙胆苦贰含量比对照高,从而为东北龙胆栽培事业的发展 以及龙胆有效成份的工业化生产提供了基础资料。     

发根农杆菌Ri质粒rol基因研究进展及在林木改良上的应用

Ri质粒上携带的诱根基因的表达不仅能导致植物被感染部位形成大量的毛根,而且由毛根容易获得再生的转化植株,这些植株可表现出许多能稳定遗传的表型变异,在植物遗传改良中有着广阔的应用前景。20世纪80年代以来,国内外学者对发根农杆菌Ri质粒及其rol基因进行了广泛深入的研究。本文重点综述了农杆碱型发根农杆菌Ri质粒的结构与功能,rol基因的位点与特征,Ri质粒的转化与rol基因的表达对植物生长发育的影响及在林木遗传改良上应用等方面的研究现状,并讨论了Ri质粒rol基因在林木遗传改良应用上存在的问题与应用前景。     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

转基因抗虫烟草研究进展

烟草为模式植物,也是外源杀虫基因最早转化成功的植物。文章从转Bt内毒素基因,植物凝集素GNA,Plec,AHA基因,蛋白酶抑制剂PIⅠ,PIⅡ,MTI,SKTI基因,昆虫特异性神经毒素基因,几丁质酶基因,畸形细胞分泌蛋白基因以及双抗虫基因等方面综述了转基因抗虫烟草的抗虫性、转基因抗虫烟草的经济性状等,展望了转基因抗虫烟草的研究和应用前景,以期对烟草害虫的治理尤其是对其他转基因抗虫作物的培育和研究有借鉴作用。     

The role of endogenous auxin in root initiation

This paper is the second part of a review which considers evidence for the involvement of auxin in root initiation. Part II examines the research being carried out with transformed plant tissues. Agrobacterium rhizogenes causes abundant root initiation at the site of inoculation. Ri plasmid T-DNA contains several genes which encode enzymes involved in the biosynthesis and metabolism of indole-3-acetic acid. Transfer of various fragments of the Ri plasmid has also been reported to confer increased sensitivity to auxin upon plant cells. Controlled expression of these genes in the plant genome potentially offer an insight for developmental plant physiologists into the role of plant growth substances in the process of root initiation. The importance of absolute levels of IAA in the stimulation of root initiation is discussed.     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

Residual phosphorus and long-term management strategies for an Ultisol

Due to their extensive growth potential, transgenic root systems arising from inoculation withAgrobacterium rhizogenes became popular in the last decade as model systems in domains as diverse as production of secondary metabolites, interactions with pathogens and symbionts, examination of gene importance in control of root development or in regulation of gene expression in roots. Wild-type bacterial strains have also been considered as useful tools to stimulate rooting on recalcitrant cuttings or microcuttings as they cause abundant root initiation at the site of inoculation.Root initiation and the in vitro growth characteristics of transformed roots result from the transfer of genes located on the root-inducing plasmid (Ri) to plant cells and their expression therein. Two sets of pRi genes are involved in the root induction process: therol (rootloci) genes located in the TL region and theaux genes of the TR region. Some of these genes being able to interact, the system appears also as a new tool to study the role of auxin in the process of root initiation. The distinctive phenotype of the transformed roots which are capable of hormone autonomous growth seems to be controlled mainly by therol genes. Theserol genes, i.e. the geneticloci rol A, rol B, rol C androl D correspond to open reading frames ORFs 10, 11, 12 and 15. In vitro experiments determined the functions of the Rol B and Rol C proteins but the functions of Rol A and Rol D are still unknown. Altered metabolism of developmental regulators or modified sensitivity to auxin have been suspected to mediate root induction and morphological abnormalities of transformed roots and plants.The target cells for transformation and the cells which are competent for root initiation will be characterized as well as the subsequent development of transgenic roots provided with various constructs from the whole T-DNA to singlerol genes. Results dealing with auxin contents in relation with root growth kinetics, phenotype and structure, will also be presented and discussed with the potential use of therol genes to control root biomass. F J de Bruijn Section editor     

Bt转基因抗虫植物研究进展

目前Bt成为世界上植物抗虫基因工程中研究和应用最多的基因。已经有多种转单一Bt和复合Bt的转基因玉米、棉花、马铃薯等作物得到大面积种植。还有许多新的具有良好抗虫性的Bt转基因植物,如水稻、大豆、油菜、苜蓿、花椰菜、蓖麻等已经试验成功,并逐渐推广种植。Bt转基因抗虫植物的培育为提高产量,减少杀虫剂的使用和保护环境做出了举足轻重的贡献。就Bt的分类、杀虫机理、Bt转基因抗虫植物的发展状况以及种植Bt抗虫植物对环境的影响进行了概述。     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

Structure and function of root-inducing (Ri) plasmids and their relation to tumor-inducing (Ti) plasmids

The abilities of Agrobacterium tumefaciens and A. rhizogenes to transform dicotyle-dons and cause crown gall and hairy root disease are caused by the presence of tumor inducing (Ti) and root inducing (Ri) plasmids. During transformation plasmid T-DNA (transferred DNA) is inserted into the plant genome. The T-region is flanked by 25 bp direct repeats, which are essential for transfer. The T-regions contain oncogenes that are expressed in the plants. Some of these code for enzymes that synthesize auxin or cytokinin. Another type, present in Ri plasmids only, appears to impose a high hormone sensitivity on the infected tissue. The T-DNA also contains genes for enzymes synthesizing opines, which the bacteria catabolize. The T-DNA transfer is initiated by the induction of genes in the virulence (vir) region of the plasmid by phenolic compounds secreted by wounded tissue. The products of the vir -genes and of chromosomal genes mediate transfer of T-DNA to the plant cells. Crown gall disease is caused by production of auxin and cytokinin by the transferred T-DNA. The T-DNA of Ri plasmids codes for at least three genes that each can induce root formation, and that together cause hairy root formation from plant tissue. Current results indicate that the products of these genes induce a potential for increased auxin sensitivity that is expressed when the transformed cells are subjected to a certain level of auxin. After this stage the transformed roots can be grown in culture without exogenous supply of hormones.