Plant transformation vector: Difference between revisions

Content deleted Content added
Sauciu1 (talk | contribs)
rewording
Citation bot (talk | contribs)
Added bibcode. | Use this bot. Report bugs. | Suggested by Abductive | Category:Molecular biology | #UCB_Category 89/577
 
(17 intermediate revisions by 10 users not shown)
Line 1:
'''Plant transformation vectors''' are [[plasmid]]s that have been specifically designed to facilitate the generation of [[transgenic plants]]. The most commonly used plant transformation vectors are termed [[binary vectors|T-DNA binary vectors]] and are often replicated in both ''[[E. coli]]'', a common lab [[Bacteria|bacterium]], and ''[[Agrobacterium tumefaciens]]'', a plant-virulent bacterium used to insert the [[recombinant (customized) DNA]] into plants.
{{Multiple issues|
{{more citations needed|date=July 2007}}
{{tone|date=May 2009}}
}}
 
Plant Transformationtransformation vectors contain three key elements;:
'''Plant transformation vectors''' are [[plasmid]]s that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed [[binary vectors|T-DNA binary vectors]] and are often replicated in both ''[[E. coli]]'', a common lab bacterium, and ''[[Agrobacterium tumefaciens]]'', a plant-virulent bacterium used to insert the recombinant (customized) DNA into plants.
Plant Transformation vectors contain three key elements;
* Plasmids Selection (creating a custom circular strand of DNA)
* Plasmids Replication (so that it can be easily worked with)
Line 11 ⟶ 7:
 
==Steps in plant transformation==
A custom DNA plasmid sequence can be created and replicated in morevarious thanways, onebut waygenerally, thisall itemmethods describesshare the generalfollowing steps.processes:
 
Plant transformation using plasmids startsbegins with the propagation of the binary vector in ''E. coli.'' When the [[bacterial culture]] reaches the appropriate density, the binary vector is isolated and purified. Then, a foreign gene can be introduced. The engineered binary vector, including the foreign gene, is re-introduced in ''E. coli'' for amplification.
 
The engineered binary factor is isolated from ''E. coli'' and is introduced into ''Agrobacteria'' containing a modified (relatively small) Ti plasmid. This engineered ''Agrobacteria'' can be used to infect plant cells. The T-DNA, containingwhich contains the foreign gene, getsbecomes insertedintegrated into athe plant cell genome. In each infected cell, the T-DNA getsis integrated at a different site in the genome.
 
The entire plant will regenerate from a single transformed cell, resulting in an organism with the transformed DNA integrated identically across all cells.
To develop a plant that carries the transformation DNA integrated in the same way in all its cells, a transformed cell is selected, from which an entire plant is regenerated.
 
=== Consequences of the insertion ===
Foreign DNA inserted
 
* Foreign DNA inserted
Insertional mutagenesis (but not lethal for the plant cell – as the organism is diploid)
 
* [[Insertional mutagenesis]] (but not lethal for the plant cell – as the organism is diploid)
Transformation DNA fed to rodents ends up in their [[phagocyte]]s and rarely other cells. Specifically this is bacterial and [[M13 bacteriophage|M13]] DNA. (This preferential accumulation in phagocytes is thought to be real and not a detection artefact since these DNA extents are thought to provoke [[phagocytosis]].) However no [[gene expression]] is known to have resulted, and this is not thought to be possible.<ref name="Goldstein-et-al-2005">{{cite journal | last1=Goldstein | first1=Daniel A. | last2=Tinland | first2=Bruno | last3=Gilbertson | first3=Lawrence A. | last4=Staub | first4=J.M. | last5=Bannon | first5=G.A. | last6=Goodman | first6=R.E. | last7=McCoy | first7=R.L. | last8=Silvanovich | first8=A. | title=Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies | journal=[[Journal of Applied Microbiology]] | publisher=[[Society for Applied Microbiology]] ([[Wiley Publishing|Wiley]]) | volume=99 | issue=1 | year=2005 | issn=1364-5072 | doi=10.1111/j.1365-2672.2005.02595.x | pages=7–23| pmid=15960661 | doi-access=free }}</ref><ref name="Lemaux-2008">{{cite journal | last=Lemaux | first=Peggy G. | title=Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I) | journal=[[Annual Review of Plant Biology]] | publisher=[[Annual Reviews (publisher)|Annual Reviews]] | volume=59 | issue=1 | year=2008 | issn=1543-5008 | doi=10.1146/annurev.arplant.58.032806.103840 | pages=771–812 | pmid=18284373}}</ref>
 
* Transformation DNA fed to rodents ends up in their [[phagocyte]]s and rarely in other cells. Specifically, this isrefers to bacterial and [[M13 bacteriophage|M13]] DNA. (This preferential accumulation in phagocytes is thought to be real and not a detection artefact since these DNA extentssequences are thought to provoke [[phagocytosis]].) However, no [[gene expression]] is known to have resulted, and this is not thought to be possible.<ref name="Goldstein-et-al-2005">{{cite journal | last1=Goldstein | first1=Daniel A. | last2=Tinland | first2=Bruno | last3=Gilbertson | first3=Lawrence A. | last4=Staub | first4=J.M. | last5=Bannon | first5=G.A. | last6=Goodman | first6=R.E. | last7=McCoy | first7=R.L. | last8=Silvanovich | first8=A. | title=Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies | journal=[[Journal of Applied Microbiology]] | publisher=[[Society for Applied Microbiology]] ([[Wiley Publishing|Wiley]]) | volume=99 | issue=1 | year=2005 | issn=1364-5072 | doi=10.1111/j.1365-2672.2005.02595.x | pages=7–23| pmid=15960661 | doi-access=free | s2cid=40454719 }}</ref><ref name="Lemaux-2008">{{cite journal | last=Lemaux | first=Peggy G. | title=Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I) | journal=[[Annual Review of Plant Biology]] | publisher=[[Annual Reviews (publisher)|Annual Reviews]] | volume=59 | issue=1 | year=2008 | issn=1543-5008 | doi=10.1146/annurev.arplant.58.032806.103840 | pages=771–812 | pmid=18284373 | bibcode=2008AnRPB..59..771L }}</ref>
 
==Plasmid selection==
A selector gene can be used to distinguish the successfully genetically bacterial modified cells from unmodified ones. AThe selector gene is integrated into the plasmid togetheralong with the desired target gene, and providesproviding the cells with resistance to an [[antibiotic]], such as [[kanamycin]], [[ampicillin]], [[spectinomycin]] or [[tetracycline]]. The desired cells, (along with any other organisms growing within the culture), can be treated with an antibiotic, allowing only the modified cells to survive. The antibiotic gene is not usually transferred to the plant cell but instead remains within the bacterial cell.
 
==Plasmids replication==
[[Plasmids]] replicate to produce many plasmid molecules in each host bacterial cell. The number of copies of each plasmid in a bacterial cell is determined by the [[replication origin]], which is the position within the plasmidsplasmid molecule where DNA replication is initiated. Most [[binary vectors]] have a higher number of plasmid copies when they replicate in ''[[E. coli]];'' however, the [[Plasmid copy number|plasmid copy-number]] is usually lesserlower when the plasmid is resident within ''[[Agrobacterium tumefaciens]]''.
Plasmids can also be replicated using the [[polymerase chain reaction]] (PCR).
 
==T-DNA region==
 
T-DNA contains two types of genes: the [[Oncogene|oncogenic genes]], encoding for [[Enzyme|enzymes]] involved in the synthesis of [[Auxin|auxins]] and [[Cytokinin|cytokinins]] and responsible for [[tumor]] formation;, and the genes encoding for the synthesis of [[Opine|opines]]. These compounds, produced by the condensation between [[Amino acid|amino acids]] and sugars, are synthesized and excreted by the crown gall cells, and they are consumed by A. tumefaciens as carbon and nitrogen sources.
 
Outside the T-DNA, are located theThe genes forinvolved thein opine [[catabolism]], the genes involved in the process of T-DNA transfer from the bacterium to the plant cell and the genes involved in[[Bacterial conjugation|bacterium-bacterium plasmid conjugative transfer]] are located outside the T-DNA.<ref (name=":0">{{Cite journal |last1=Hooykaas and|first1=Paul J. J. |last2=Schilperoort, |first2=Rob A. |date=1992;-05-01 Zupan|title=Agrobacterium and Zambrysky,plant genetic engineering |url=https://doi.org/10.1007/BF00015604 |journal=Plant Molecular Biology |language=en |volume=19 |issue=1 |pages=15–38 |doi=10.1007/BF00015604 |pmid=1600167 |bibcode=1992PMolB..19...15H |s2cid=36172990 |issn=1573-5028|url-access=subscription }}</ref><ref name=":1">{{Cite journal |last1=Zupan |first1=J. R. |last2=Zambryski |first2=P. |date=1995)-04-01 |title=Transfer of T-DNA from Agrobacterium to the Plant Cell |url=https://doi.org/10.1104/pp.107.4.1041 |journal=Plant Physiology |volume=107 |issue=4 |pages=1041–1047 |doi=10.1104/pp.107.4.1041 |issn=0032-0889 |pmc=157234 |pmid=7770515}}</ref> The T-DNA fragment is flanked by 25-bp direct repeats, which act as a cis -element signal for the transfer apparatus. The process of T-DNA transfer is mediated by the cooperative action of [[Protein|proteins]] encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The Ti plasmid also contains the genes for opine catabolism produced by the crown gall cells and regions for conjugative transfer and for its own integrity and stability. The 30 kb virulence (vir) region is a [[regulon]] organized in six [[Operon|operons that are]] essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE).<ref (Hooykaasname=":0" and/><ref Schilperoort,name=":1" 1992;/><ref>{{Cite Zupanjournal and|last1=Jeon Zambryski,|first1=Geoung-A 1995,|last2=Eum Jeon|first2=Jin-seong et|last3=Sim al.,|first3=Woong Seop |date=1998-02-01 |title=The Role of Inverted Repeat (IR) Sequence of the virE Gene Expression in Agrobacterium tumefaciens pTiA6. Different|journal=Molecules and Cells |volume=8 |issue=1 |pages=49–53 |doi=10.1016/S1016-8478(23)13391-7 |pmid=9571631 |issn=1016-8478|doi-access=free }}</ref> Several chromosomal-determined genetic elements have shown their functional role in the attachment of ''A. tumefaciens'' to the plant cell and bacterial colonization:. theThe loci chvA and chvB, are involved in the synthesis and excretion of the b -1,2 [[glucan]],<ref>{{Cite (journal |last1=Cangelosi et|first1=G al.A |last2=Martinetti |first2=G |last3=Leigh |first3=J A |last4=Lee |first4=C C |last5=Theines |first5=C |last6=Nester |first6=E W |date=March 1989 |title=Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan |journal=Journal of Bacteriology |language=en |volume=171 |issue=3 |pages=1609–1615 |doi=10.1128/jb.171.3.1609-1615.1989) |issn=0021-9193 |pmc=209788 |pmid=2921245}}</ref> the {{not a typo|chvE}} required for the sugar enhancement of vir [[Gene induction|genes induction]] and [[Bacterial chemotaxis - general|bacterial chemotaxis]].<ref>{{Cite (journal |last1=Ankenbauer et|first1=R alG |last2=Nester |first2=E W |date=November 1990 |title=Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides |journal=Journal of Bacteriology |language=en |volume=172 |issue=11 |pages=6442–6446 |doi=10.,1128/jb.172.11.6442-6446.1990 |issn=0021-9193 |pmc=526831 |pmid=2121715}}</ref><ref>{{Cite journal |last1=Cangelosi |first1=G A |last2=Ankenbauer |first2=R G |last3=Nester |first3=E W |date=September 1990, |title=Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. |journal=Proceedings of the National Academy of Sciences |language=en |volume=87 |issue=17 |pages=6708–6712 |doi=10.1073/pnas.87.17.6708 |doi-access=free |issn=0027-8424 |pmc=54606 |pmid=2118656|bibcode=1990PNAS...87.6708C }}</ref><ref name=":2">{{Citation |last1=Cangelosi et|first1=Gerard alA., 1990,|title=Bacterial Genetic Systems |date=1991 |pages=384–397 |url=https://doi.org/10.1016/0076-6879(91)04020-o the|access-date=2024-03-09 |publisher=Elsevier |doi=10.1016/0076-6879(91)04020-o |last2=Abest |first2=Elaine |last3=Martinetti |first3=Gladys |last4=Nester |first4=Eugene W.|chapter=Genetic Analysis of Agrobacterium |series=Methods in Enzymology |volume=204 |pmid=1658565 |isbn=978-0-12-182105-0 |url-access=subscription }}</ref> The cell locus, is responsible for the synthesis of [[cellulose]] fibrils.<ref>{{Cite (journal |last=Matthysse |first=A G |date=May 1983); the|title=Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection |journal=Journal of Bacteriology |language=en |volume=154 |issue=2 |pages=906–915 |doi=10.1128/jb.154.2.906-915.1983 |issn=0021-9193 |pmc=217544 |pmid=6302086}}</ref> The {{not a typo|pscA (exoC)}} locus playingis its roleinvolved in the synthesis of both cyclic glucan and acid [[Glycan|succinoglycan]].<ref>{{Cite (journal |last1=Cangelosi et|first1=G at.A |last2=Hung |first2=L |last3=Puvanesarajah |first3=V |last4=Stacey |first4=G |last5=Ozga |first5=D A |last6=Leigh |first6=J A |last7=Nester |first7=E W |date=May 1987, 1991)|title=Common loci for Agrobacterium tumefaciens and theRhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions |journal=Journal of Bacteriology |language=en |volume=169 |issue=5 |pages=2086–2091 |doi=10.1128/jb.169.5.2086-2091.1987 |issn=0021-9193 |pmc=212098 |pmid=3571162}}</ref><ref name=":2" /> The att locus, which is involved in the cell [[Surface protein|surface proteins]].<ref>{{Cite (journal |last=Matthysse, |first=Ann G. |date=October 1987) |title=Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells |journal=Applied and Environmental Microbiology |language=en |volume=53 |issue=10 |pages=2574–2582 |doi=10.1128/aem.53.10.2574-2582.1987 |issn=0099-2240 |pmc=204148 |pmid=16347473|bibcode=1987ApEnM..53.2574M }}</ref>
 
==References==