发根农杆菌介导的药用植物遗传转化研究进展

发根农杆菌介导的植物遗传转人旨近年来发展起来的一项新的植物遗传转化技术。本文就发根农杆菌Ｒ１闰的结构、发根农杆菌介导的植物遗传转化的方法和步骤,以及运用这一技术获得药用植物次生代谢产物的研究进展作一全面介绍。     

发根农杆菌介导的药用植物遗传转化研究

利用发根农杆菌(Agrobacterium rhizogenes)诱导药用植物产生的毛状根具有生长迅速,合成能力强和遗传性稳定等优点,已成为一种新的培养系统。就影响发根农杆菌介导的药用植物遗传转化的因素作一概述。     

高等植物在发根农杆菌介导下的遗传转化

本文介绍了发根农杆菌的生物学特性、遗传转化的操作技术、RiT-DNA转化体的形态特征以及发根农杆菌转化的利用。与根癌农杆菌不同,农杆碱型发根农杆菌的RiT-DNA含有生长素合成基因、农杆碱、甘露碱合成基因,不含有细胞分裂素合成基因,它的转化体首先是转化根。发根农杆菌介导的植物遗传转化,在次生代谢产物生产,植物抗逆性育种以及细菌与植物进化关系的研究等方面具有广泛的应用前景。     

超声波辅助处理对发根农杆菌介导的苦豆子遗传转化的影响

以发根农杆菌转化苦豆子的子叶和下胚轴外植体,结果表明,辅助以超声波处理有助于转化率的提高,当超声波(功率120W,震荡频率50kHz)处理25min时,转化率达到最高峰(子叶为83．7％,下胚轴为39．1％),分别高于对照(14．4％和9．8％)69．3％和29．3％。在此条件下,进一步添加AS l00μmol/L,可使转化率再进一步提高9．9％和7．6％。     

农杆菌介导的玉米遗传转化研究进展

本文就农杆菌介导的玉米遗传转化的技术要点及原理等进行了综述,并对各种影响农杆菌率玉米效率的关键因子包括农杆菌的菌株与载体、标记基因、受体材料的基因型、来源和发育状态以及组织培养的条件等进行了讨论。     

发根农杆菌Ri质粒在药用植物生物工程中的应用

发根农杆菌(Ａｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓ)与根癌农杆菌(Ａ.ｔｕｍｅｆａｃｉｅｎｓ)属于根瘤菌科,均为革兰氏阴性菌[1]。它们在侵染植物后引起的症状不同,含有Ｔｉ质粒的根癌农杆菌在侵染植物后形成冠瘿瘤(ｃｒｏｗｎｇａｌｌ),而含有Ｒｉ质粒的发根农杆菌表现与其不同,它在感染植物后在植物的伤口部位诱发产生毛状根(ｈａｉｒｙｒｏｏｔ)。由发根农杆菌侵染植物诱导产生的毛状根具有生长快、分枝多、根毛多等特点。发根农杆菌Ｒｉ质粒与根癌农杆菌Ｔｉ质粒结构相似,都具有高效率转移的Ｔ-ＤＮＡ区和致病的Ｖｉｒ区…     

农杆菌介导的墨兰遗传转化

转基因育种是快速定向改良兰花育种目标性状的有效方法,但迄今未见有关墨兰转基因育种的研究报道。试验以‘企剑白墨’墨兰Cymbidium sinensis cv.‘Qijianbaimo’的根状茎为受体材料,研究了影响农杆菌介导墨兰遗传转化效率的因素,以建立有实用价值的墨兰遗传转化技术体系。结果表明,受体的预培养时间、乙酰丁香酮的添加方式及浓度、农杆菌工程菌液浓度(OD600)、侵染时间和共培养时间均对‘企剑白墨’根状茎的GUS瞬时表达率有显著影响。以预培养39 d的根状茎尖为材料,在添加200μmol/L乙酰丁香酮,OD600为0.9的工程菌液中侵染35 min后,转入添加200μmol/L乙酰丁香酮的共培养基中培养7 d时,‘企剑白墨’根状茎的GUS瞬时表达率最高,为11.67%。采用上述条件对‘企剑白墨’墨兰进行遗传转化,经PCR鉴定和GUS染色检测,从400株再生植株中获得了3株转基因植株,转化率为0.75%。研究表明通过农杆菌介导法对墨兰进行遗传改良是可行的。     

乙酰丁香酮对发根农杆菌遗传转化黄瓜的影响

乙酰丁香酮对发根农杆菌遗传转化黄瓜的影响施和平李玲潘瑞炽（华南师范大学生物系,广州５１０６３１）ＥＦＦＥＣＴＯＦＡＣＥＴＯＳＹＲＩＮＧＯＮＥＯＮＴＲＡＮＳＦＯＲＭＡＴＩＯＮＯＦＣＵＣＵＭＩＳＳＡＴＩＵＳＢＹＡＧＲＯＢＡＣＴＥＲＩＵＭＲＨＩＺＯＧＥＮＥ...     

The Genetic Transformation of Pueraria as Medicinal Plant by Agrobacterium rhizogenes

Pueraria radix (the dried root of Pueraria plant) is known as a traditional Chinese drug. Hairy roots of Pueraria lobata (Willd.) Ohwi, P.lobata var. montana and P. phaseoloides (Roxb.) Benth. transformed by Agrobacterium rhizogenes (Riker et al .) Conn R1601 were developed directly from the surface of sterile leaves in vitro . The transformation frequency was 16.6%, 16.2% and 26.6%, respectively. All hairy roots in the three species displayed the typical phenotypes of rapid growth, highly branched and plagiotropism, and also exhibited hormone autotrophy and resistance to kanamycin.The genetic transformations were confirmed by opine paper electrophoretic analysis, rol gene PCR amplification and molecular hybridization.     

发根土壤杆菌对葛属药用植物的遗传转化

葛根是传统中药。利用发根土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒｈｉｚｏｇｅｎｅｓ （Ｒｉｋｅｒ ｅｔ ａｌ．）Ｃｏｎｎ）Ｒ１６０１转化野葛（Ｐｕｅｒａｒｉａ ｌｏｂａｔａ（Ｗｉｌｌｄ．）Ｏｈｗｉ）、山葛（Ｐ．ｌｏｂａｔａ ｖａｒ．ｍｏｎｔａｎａ）和三裂叶野葛（Ｐ．ｐｈａｓｅｏｌｏｉｄｅｓ（Ｒｏｘｂ．）Ｂｅｎｔｈ．）离体叶片,在叶片表面直接形成毛状根。毛状根的诱导频率分别为１６．６％、１６．２％和     

Genetic Transformation of Cucumis sativus by Agrobacterium rhizogenes

Hairy roots were obtained in vitro 10 days after inoculation of cucumber ( Cucumis sativus L. ) cotyledon explants with the strains of Agrobacterium rhiwgenes R1000 and R1601. The frequency of the cotyledon explants transformed by R1000 and R1601 was up to 87.5% and 88.9%, respectively. All hairy roots induced by the strains of R1000 and R1601 grew rapidly on solid hormone-free MS medium. The roots incited by A. rhizogenes R1000 could be divided into three phenotypes. The roots of phenotype Ⅰ were similar to the normal ones, but had more numerous lateral roots. Roots of phenotype m were much stouter and shorter, they elongated very slowly and were more highly branched than roots of phenotype Ⅰ . Roots of phenotype Ⅱ were of intermediate in appearance. However, the roots incited by A. rhizogenes R1601 appeared similar to phenotype Ⅰ roots incited by A. rhizogenes R1000. Transformation was confirmed by opine detection.     

中药植物黄山药发根基因的遗传转化

以发根农杆菌菌株A4转染已预培养1d的黄山药茎段后,共感染3d,其转化效果最佳;转化毛状根在无生长调节物质的MS培养基上培养可获得丛状芽,并发育成植株。     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

发根农杆菌A4菌株转化苜蓿悬浮培养物

将苜蓿无菌苗下胚轴切割后,在附加２ｍｇ／Ｌ２,４Ｄ的ＭＳ培养基上诱导愈伤组织。愈伤组织在附加０５ｍｇ／Ｌ２,４Ｄ的ＳＨ培养基中悬浮培养。悬浮培养物在用于转化之前,用０４５ｍｏｌ／Ｌ甘露醇处理１ｈ,然后用０１６ｍｏｌ／ＬＣａＣｌ２·２Ｈ２Ｏ洗涤两次。预处理后的悬浮培养物用ＳＨ培养基悬浮（１０ｍｌ／ｇ悬浮培养物）,再加０２ｍｌ农杆菌悬浮液,于２５±２℃共培养２ｄ。共培养的悬浮培养物洗涤后在附加０５ｍｇ／Ｌ羧苄青霉素的无激素培养基上选择培养。悬浮培养天数、悬浮培养基激素组成和选择培养基种类明显影响转化频率。纸电泳分析表明７０％的转化体可以合成农杆碱和甘露碱。染色体观察显示转化组织细胞存在严重的数目和结构变异     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

发根农杆菌介导药用甘薯西蒙1号的遗传转化

用发根农杆菌A4分别感染药用甘薯西蒙1号的叶片、茎切段、叶柄等外植体,诱导出毛状根,并对毛状根进行了离体培养.采用L9(34)正交设计法优化甘薯西蒙1号的毛状根诱导条件;PCR扩增检测转化毛状根;用高效液相色谱仪检测了毛状根中咖啡酸的含量.结果表明:转化中茎切段是最合适的外植体,最佳感染时间20 min,共培养最佳时间为2天;PCR扩增检测表明发根农杆菌Ri质粒的T-DNA片段已整合进植物的基因组中;经高效液相色谱仪证实毛状根中含有咖啡酸,含量为0.03792 mg/g.     

Induction of Pseudoactinorhizae by the Plant Pathogen Agrobacterium rhizogenes

Infection of Elaeagnus angustifolia cotyledonary wounds by Agrobacterium rhizogenes strain NCPPB 2659 resulted in the formation of pseudoactinorhizae on roots differentiated from callus. These pseudoactinorhizal root nodules were anatomically indistinguishable from the actinorhizae induced by the plant's microsymbiont Frankia. This unusual hairy root phenotype provides support for the concept that the genetic program for actinorhiza morphogenesis resides in the plant's genome.