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…     

发根农杆菌Ri质粒rolB基因研究进展（综述）

RiT-DNArolB基因是发根农杆菌转化植物的决定因子,rolB基因表达引起转基因植物形成大量毛状根（hairy root)。本文介绍近年来rolB基因的位点,表达与调控及RolB蛋白结构与功能方面的研究进展。     

Potato transformation byAgrobacterium rhizogenes Ri plasmid

Potato root cultures transformed by_pRiA4 T-DNA do not regenerate plants in vitro spontaneously, but regeneration can be induced by a sequence of callogenesis and regeneration media. Both T_l and T_r-DNA were found by Southern blotting. Regenerated transformants showed characteristic morphological deviations in the in_vitro cultures. Plants in the soil showed differences in the inflorescence and tuber development.     

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.     

发根农杆菌Ri质粒及其在植物科学中的应用

阐述了发根农杆菌的性质及Ri质粒的结构和功能,介绍了毛状根的诱导及其鉴定方法,并对Ri质粒在植物基因工程、植物次生代谢产物生产、植物品种改良和植物栽培等方面的应用进行了综述。     

Identification of hairy root loci in the T-regions of Agrobacterium rhizogenes Ri plasmids

Summary Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the T_L-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the T_L-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the T_L-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the T_L-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the T_L-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114     

Characterization of the virA gene of the agropine-type plasmid pRiA4 of Agrobacterium rhizogenes

We sequenced a 4.2-kb DNA region encompassing the vir A locus of the hairy-root-inducing plasmid pRiA4, and compared its sequence with the published vir A region sequences of four tumor-inducing plasmids. An open reading frame capable of coding for 829 amino acids was identified for vir A. Deletion mutants of vir A constructed by fusing to lacZ, but not the wild-type game itself, were efficiently expressed in Escherichia coli when they were put downstream front the lac promoter. These fused gene products became soluble or insoluble depending on the length of their lacZ moieties.     

Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes

Abstract Efficient transformation of strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes by electroporation with binary Ti plasmid vector is reported. This procedure yields rates of transformation of 10⁶-10³ per μg DNA, which is several orders of magnitude greater than previously published procedures for this genus, the efficiency of transformation varies with the bacterial strain used. This procedure will be useful for the construction of plant DNA libraries directly in Agrobacterium .     

Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid of Agrobacterium

The symbiotic plasmid of Rhizobium trifolii G1008 was mobilized to other Rhizobium strains and to Agrobacterium using Tn5-Mob, a transposon that confers on a host replicon the ability to be mobilized in trans by RP4. Incompatibility was observed between pSymG1008 and the hairy-root-inducing plasmid pRi1855. Agarose gel electrophoresis revealed that pRi1855 was eliminated as an autonomous element in the presence of pSymG1008 and its absence was correlated with loss of the ability to induce hairy root disease. This indicates a close ancestral relationship between a Rhizobium symbiotic plasmid and a plant pathogenic plasmid of Agrobacterium. pSymG1008 and pRi1855 can be assigned to the IncRh-3 incompatibility group. Furthermore, pSymG1008 was mobilized at low frequency to R. phaseoli 51E and the transconjugants isolated had lost the indigenous Sym plasmid and the ability to nodulate beans.     

发根农杆菌Ri质粒rol基因研究的综述

发根农杆菌Ｒｉ质粒ｒｏｌ基因研究的综述①周跃钢王三根（西南农业大学生理生化教研室,重庆４００７１６）ＲｅｖｉｅｗｏｆＳｔｕｄｉｅｓｏｎＲｏｌＧｅｎｅｓｏｆＲｉＰｌａｓｍｉｄｓｆｒｏｍＡｇｒｏｂａｃｔｅｒｉｕｍｒｈｉｚｏｇｅｎｅｓＺｈｏｕＹｕｅｇａｎｇ...     

Genome structure of Ri plasmid (3). Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes.

The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced. The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5. The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids. We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.     

Hairy root: plasmid encodes virulence traits in Agrobacterium rhizogenes

Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10(6) daltons), pAr15834b (154 x 10(6) daltons), and pAr15834c (258 x 10(6) daltons). Kanamycin-resistant transconjugants were selected in a cross of kanamycin-resistant derivate of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plasmids from virulent transconjugants during growth at 37 degrees C indicated that virulence genes reside on pAr15834b, whereas agrocin 84 sensitivity genes reside on pAr15834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain of A. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pAr15834c is a cointegrate of pAr15834a and pAr15834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pAr15834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.