Vectors and its types

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Vectors and its types

  • A vector is a circular DNA molecule capable of independent existence and replication as example plasmid and Virus.
  • In case of plants agrobacterium plasmid are the most commonly used vectors.
  • A tumefacieus has the ti plasmid while a reasons has the ri plus made this plasmid have similar general feature and can I interchange between the two species.
  • Both the plasmid have risen which content jeans for or metabolism and phytohormone independence this region is transferred into The plant cell and is integrated into their genome.
  • They have another reason called vir region which produce among other things a endonuclease essential for the exhibition transfer of the reason into plant cell.
  • The genes to the transferred are placed within t-dna of this plasmid.
  • Therefore this transformation of vector into plants two vectors are prepared by genetic modification.
  • They are co integrated vector and binary vector.

Co integrated vector

  • In the CO integrated vector gene transferred is integrated with in the t DNA of a resident ti plasmid forming and intermediate vector.
  • These are also known as hybrid ti-plasmid.
  • These factors were among the first types of modified and engineered.
  • TI plasmid devised for agrobacterium mediated transformation but they are not widely used now a days.
  • This vectors are constructed by homologous recombination of a bacterial plasmid with the t DNA reason of an endogenous ti plasmid in agrobacterium.
  • Integration of the two plasmid requires a reason of homologous present in both.
  • These vectors are necessary in this system:

Disarmed agrobacterium ti plasmid:

  • In this ti plasmid the oncogenes located in the t DNA reason have been replaced by exogenous DNA.
  • These vectors include:-

Sev series:-

  • The right border of the t DNA together with the phytohormone genes coding for cytokinin and oxygen are removed and replaced by a bacterial kanamycin resistance gene while the left border and a small part of the segment of the original t DNA referred to as left inside homology are left intact.

PGV SERIES:

  • The phytohormone genes are excised and substituted by part of pbr322 vector sequence.
  • The left and right border sequence as well as the nopeline synthase gene of the ti plasmid covered.

Intermediate vectors:

  • These are small pbr322 based plasmid containing the t DNA reason.
  • They are used to overcome the problems derived from the large size of disarmed ti plasmid and their lake of unique restriction sites.
  • Intermediate vectors are replicated in e coli and transferred into agrobacterium by conjugation.
  • They can not replicate in a tumefaciens and therefore carry DNA segments homologous to the disarmed T DNA to permit recombination to form a CO integrated t DNA structure.

Helper vectors:

  • These are small plasmids maintained in e coli that contain transfer and mobilization gene which allowed the transfer of the consideration deficient intermediate vectors into agrobacterium.
  • Resulting co integrated plasmid made assembled by vitro manipulation normally contents:
  • The vir genes
  • The left and right t-DNA borders
  • An exogenous DNA sequence between the two T DNA borders.
  • Plant and bacterial selectable markers.
  • Although co integrated vectors have been designed to allow site specific recombination based on the recombination system of the phase P1.
  • Large size of CP Plus made it is very difficult to handle and also presence of oncogenes or tumors carrying genes.
  • Therefore co integrate vector is engineered genetically to insert into the plant.

In this strategy both the t DNA with our gene of interest and we are reason are present in the same tractor used for transformation.

Vectors

Making of co integrated vectors

  • At first and intermediate vector is mad using e coli plasmid + reason + t DNA borders + origin of replication + pbr322 sequences.
  • Second vector is a disarmed PT vector =gene of interest + some markers + pbr322 sequences.
  • Both intermediate vector and disarmed has some sequence in common which is PBR 322 sequences.
  • Therefore by homologous recombination co integration of two plasmid will take place within agrobacterium.
  • Now we have a CO integrated vector that has the t DNA with our gene of interest within the t DNA borders and VIR region.

Binary vector: Transformation of tobacco plant using co integrated vector:-

  • The binary vector system consists of 2 plasmid that are involved in gene transformation and both are able to replicate separately inside the agrobacterium cell.
  • The first plasmid is a modified ti plasmid which provides the VIR functions in trance and second contain gene of interest between the t DNA borders.
  • Disarmed helper ti plasmid have been engineered by removing the oncogenic jeans while still providing the necessary VIR gene product required for transferring the t DNA to the host plant cell.
  • A pair of plasmid consists of binary plasmid and helper plasma and are used to produce genetically modified plants.
  • They are artificial vectors that have been created from the naturally occurring ti plasmid found in agrobacterium tumefaciens.
  • The binary vector is a shuttle vector so called because it is able to replicate in multiple hosts.
  • System in which t DNA and genes are located on a separate replicons are called t-DNA binary system.
  • t DNA is located on the binary vectors.
  • the replicon containing the VIR genes become known as VIR helper.
  • There are several binary vector systems that differ mainly in the plasmid region that facilitates replication in agrobacterium.
  • Commonly used binary vectors include PBINI9, PBVP, P GREEN.

Vectors

Development of binary vectors:

  • The PBINI9 plasmid was developed in the 1980 and is one of the first and most widely used to binary vector plasmid.
  • P GREEN plasmid which was developed in 2000 is a newer version of the binary vectors that allow for choice of promoters selectable markers and reporter genes.
  • Another distinguish feature of P GREEN plasmid is it’s large reduction in size PBINI9 there for increasing its transformation efficiency.
  • Along with higher transformation efficiency P green has been engineered to ensure your transformation integrity.
  • Due to polarity difference in the left and right borders the right border of the t DNA enters the host plant first.
  • If the selectable marker is near the right border and the transformation process is interrupted the resulting plant may have expression of a selectable marker but contain no t-DNA giving of false positive.
  • The p green plasmid has the selectable marker entering the host last so that any expression of the marker will result in full transgene integration.

Vectors