scholarly journals A Second T-Region of the Soybean-Supervirulent Chrysopine-Type Ti Plasmid pTiChry5, and Construction of a Fully Disarmed vir Helper Plasmid

2000 ◽  
Vol 13 (10) ◽  
pp. 1081-1091 ◽  
Author(s):  
Karuppaiah Palanichelvam ◽  
Philippe Oger ◽  
Steven J. Clough ◽  
Chung Cha ◽  
Andrew F. Bent ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Ti Plasmid ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Selection For ◽  
Border Repeats ◽  
The Right ◽  
Binary Plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadori opines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 coinherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

scholarly journals Agrobacterium tumefaciens-MEDIATED IN PLANTA TRANSFORMATION METHOD FOR THE SoSPS1 GENE IN CITRUS PLANTS (Citrus nobilis L.)

2020 ◽  
Vol 7 (1) ◽  
pp. 31
Author(s):  
Ni Putu Ayu Erninda Oktaviani Suputri ◽  
Rindang Dwiyani ◽  
Ida Ayu Putri Darmawanti ◽  
Bambang Sugiharto
Keyword(s):  
Tissue Culture ◽  
Agrobacterium Tumefaciens ◽  
Cauliflower Mosaic Virus ◽  
Total Soluble Protein ◽  
35S Promoter ◽  
Nptii Gene ◽  
Main Function ◽  
In Planta Transformation ◽  
In Planta ◽  
Regeneration System

The SoSPS1 gene of sugar cane plants previously subjected to Agrobacterium tumefacienmediated cloning was to be transferred to citrus plants to increase metabolism of sucrose in plant. The T-DNA harbored the SoSPS1 gene under the control of the CaMV 35S promoter from the cauliflower mosaic virus and contained the NPTII gene (kanamycin resistance gene) as a selectable marker for transformant selection. Generally, gene transformation in plants is carried out by tissue culture. However, tissue culture has several disadvantages such as its being time-consuming, its sometimes resulting in somatic mutations and somaclonal variations, and the requirement of sterile conditions in the procedure of gene transfer. In planta transformation is a useful system for those plants that lack tissue culture and regeneration system. The main function of in planta transformation is to recover the advantages of tissue culture as an efficient, quick method, including its ability to produce a large number of transgenic plants and to accumulate a high concentration of total soluble protein in short time. There are two procedures of in planta transformation for the seeds of citrus plants, namely “prick and coat” and “seed tip-cutting and imbibition”. In the prick and coat method, seeds are pricked on their entire surfaces and smeared with a suspension of Agrobacterium tumefaciens. In the seed tip-cutting and imbibition method, on the other hand, seeds are cut at the tip and soaked in a suspension of Agrobacterium tumefaciens. The leaves derived from seeds treatment were taken as samples for DNA extraction and PCR using primers of the NPTII gene (Forward: 5’-GTCATCTCACCTTCCTCCTGCC-3’; Reverse: 5’-GTCGCTTGGTCGGTCATTTCG-3’). This research found that only the seed tip-cutting and imbibition plants amplified along the 550-bp band, while those of the prick and coat method did not. Additionally, the T-DNA was successfully integrated into the genome of the plants treated with the seed tip-cutting and imbibition method but not with the prick and coat.

Agrobacterium tumefaciens -mediated transformation of tomato (Solanum lycopersicum)

2017 ◽  
Vol 6 (6) ◽  
Author(s):  
Shanthala Mallikarjunaiah ◽  
Sowmya Moudgalya
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plasmid Dna ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Alkaline Lysis ◽  
Annexin Gene ◽  
Pcr Method

The present investigation describes the development of genetically engineered tomato plants with annexin gene. The alkaline lysis method is used to isolate the plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) from E.coli (DH5α strain). The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

Agrobacterium tumefaciens -mediated transformation of tobacco (Nicotiana tabaccum)

2017 ◽  
Vol 6 (4) ◽  
Author(s):  
Shanthala Mallikarjunaiah ◽  
Mahesh Pattabhiramaiah ◽  
Chethan Gowda
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plasmid Dna ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Nptii Gene ◽  
Helper Plasmid ◽  
Alkaline Lysis ◽  
Annexin Gene ◽  
Pcr Method

The present study involves the development of genetically engineered tobacco plants with annexin gene. The plasmid DNA of pUC 19 vector having the desired Annexin gene (3Kbp) and the plasmid DNA of the binary vector pGPTV (13 Kbp) were isolated from E.coli (DH5α strain) by alkaline lysis method. The purified pUC 19/Annexin and pGPTV plasmid were restriction digested using the restriction enzymes EcoRI and XbaI as a linearised band was eluted from the gel. The digested plasmid shown in the band pattern in the gel were cut by gel elution technique and purified from other reaction mixture. Then the dot spot test was done to calculate the concentration of pGPTV and Annexin gene. The recombinant PGPTV plasmid with the annexin gene in Agrobacterium tumefaciens MTCC 431 was mobilized and transferred to plant system through the mobilization helper plasmid pRK2013. The kanamycin resistance gene (NPT II) was used as a selective marker. The calli used for isolating the genomic DNA which was then amplified for confirmation of annexin gene. The nptII gene of 800 bp serves as a selectable marker system in plants and its amplification confirmed the presence of annexin gene in transgenic plants by PCR method.

scholarly journals Overexpression of the Golden SNP-Carrying Orange Gene Enhances Carotenoid Accumulation and Heat Stress Tolerance in Sweetpotato Plants

Antioxidants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 51
Author(s):  
So-Eun Kim ◽  
Chan-Ju Lee ◽  
Sul-U Park ◽  
Ye-Hoon Lim ◽  
Woo Sung Park ◽  
...  
Keyword(s):  
Heat Stress ◽  
Stress Tolerance ◽  
Ipomoea Batatas ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Cauliflower Mosaic Virus ◽  
Carotenoid Content ◽  
35S Promoter ◽  
Storage Roots ◽  
Heat Stress Tolerance

Carotenoids function as photosynthetic accessory pigments, antioxidants, and vitamin A precursors. We recently showed that transgenic sweetpotato calli overexpressing the mutant sweetpotato (Ipomoea batatas [L.] Lam) Orange gene (IbOr-R96H), which carries a single nucleotide polymorphism responsible for Arg to His substitution at amino acid position 96, exhibited dramatically higher carotenoid content and abiotic stress tolerance than calli overexpressing the wild-type IbOr gene (IbOr-WT). In this study, we generated transgenic sweetpotato plants overexpressing IbOr-R96H under the control of the cauliflower mosaic virus (CaMV) 35S promoter via Agrobacterium-mediated transformation. The total carotenoid contents of IbOr-R96H storage roots (light-orange flesh) and IbOr-WT storage roots (light-yellow flesh) were 5.4–19.6 and 3.2-fold higher, respectively, than those of non-transgenic (NT) storage roots (white flesh). The β-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, IbOr-R96H plants showed greater tolerance to heat stress (47 °C) than NT and IbOr-WT plants, possibly because of higher DPPH radical scavenging activity and ABA contents. These results indicate that IbOr-R96H is a promising strategy for developing new sweetpotato cultivars with improved carotenoid contents and heat stress tolerance.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

2012 ◽  
Vol 39 (9) ◽  
pp. 764 ◽  
Author(s):  
Gi-Ho Lee ◽  
Seong-Han Sohn ◽  
Eun-Young Park ◽  
Young-Doo Park
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Histone Deacetylase 1 ◽  
Transgenic Lines ◽  
Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours

1980 ◽  
Vol 144 (3) ◽  
pp. 353-376 ◽  
Author(s):  
M. Lemmers ◽  
M. De Beuckeleer ◽  
M. Holsters ◽  
P. Zambryski ◽  
A. Depicker ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Crown Gall ◽  
Ti Plasmid ◽  
Internal Organization ◽  
Organization Boundaries

scholarly journals Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 737-748
Author(s):  
D K Asch ◽  
G Frederick ◽  
J A Kinsey ◽  
D D Perkins
Keyword(s):  
Neurospora Crassa ◽  
Plasmid Dna ◽  
Repetitive Sequence ◽  
Single Copy ◽  
The Other ◽  
Chromosomal Dna ◽  
End Joining ◽  
Dehydrogenase Gene ◽  
The Right ◽  
Complete Deletion

Abstract We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

1996 ◽  
Vol 26 (6) ◽  
pp. 909-917
Author(s):  
Madoka Gray-Mitsumune ◽  
Bong Y. Yoo ◽  
Pierre J. Charest
Keyword(s):  
Fusion Gene ◽  
Black Spruce ◽  
Gus Activity ◽  
Chimeric Gene ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Zygotic Embryos ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Rbcs Promoter

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

Analysis of the results of competitions held in 2020 for the right to receive grants from the President of the Russian Federation for state support of young Russian scientists and competitive selection for personal scholarships named after ZH.I. Alferov for young scientists in the field of physics and nanotechnology

2021 ◽  
pp. 44-51
Author(s):  
B.V. Ivanov ◽  
S.V. Kristalinskaya ◽  
E.A. Gladysheva ◽  
D.A. Dobrynin
Keyword(s):  
Russian Federation ◽  
State Support ◽  
Competitive Selection ◽  
Research Areas ◽  
Federal Districts ◽  
The Russian Federation ◽  
Selection For ◽  
Number Of Publications ◽  
The Right ◽  
To Receive

The article presents the results of the analysis of the indicators of the competitions of grants of the President of the Russian Federation held in 2020 for state support of young Russian scientists and competitive selection for receiving personal scholarships named after J.I. Alferov for young scientists in the field of physics and nanotechnology: generalized data on the number of publications of winners, distribution of participants and winners by research areas, federal districts, regions, departments and organizations.

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