PLANT VECTORS REKHA PULICHERLA. Plasmids A circular piece of DNA that is not a part of the...

PLANT VECTORS

REKHA PULICHERLA

PlasmidsA circular piece of DNA that is not a part of the bacteria’s genome. It is ‘extra-chromosomal.’

Analogy: The Plasmid is the little dog, and the Chromosomal DNA is the big dog. (The cell cannot live without the big dog however…)

• Carrier for DNA during the recombinant DNA process.

• Plasmid-piece of free-floating DNA in the cytoplasm of bacteria.

• Double-stranded, circular molecules that replicate independently of the chromosome.

• Common features of eukaryotic vectors include

Most vectors carry marker genes that allow the recognition of transformed cells

Others die due to the action of an antibioticThey are called as selectable markers

Kanamycin resistance used for selection of transformants

• The other features of vectors include1.Multiple unique restriction sites2.Bacterial origin of replication

• Agrobacterium Ti plasmid is the most preferred as it has a wide range of host range and capacity to transfer genes due to T-DNA border sequences

Ti &Ri Plasmids• Most commonly used vectors in plant systems

• Ti plasmid is found in tumor inducing strains of A.tumefaciens

• Ri plasmid is found in root inducing (hairy root disease) strains of A.rhizogenes

• T-DNA is a part of both the plasmids

Ti Plasmids

• Ti plasmids have 4 regions in common1.Region A comprising the T (transferred)-DNA

responsible for tumor induction2.Region B responsible for replication3.Region C responsible for conjugation4.Region D responsible for virulence called as vir

(virulence) region

T-DNA Regions1. An onc (oncogenecity) region consisting of 3

genes- tms1, tms2 (shooty locus) tms2 (rooty locus)

• This region is responsible for synthesis of 2 phytohormones namely

IAA (indole acetic acid-auxin) IPA (isopentyladenosine monophosphate-

cytokinin)

• These genes encode enzymes for synthesis of the hormones

• Their incorporation in the plant genomes lead to synthesis in host plant

• This in turn alter the host development leading to formation of crown gall

2. An os region responsible for synthesis of unusual amino acids or sugar derivatives called as opines

• Opines are derived from variety of compounds found in plant cells

• E.g. – octopine and nopaline• The synthesis of these two enzymes are coded

by T-DNA• Depending whether Ti plasmid encodes

octopine or nopaline it is described as octopine type Ti plasmid or nopaline type Ti plasmid

• Ti plasmid carry genes that can catabolize opines which are utilized as source of carbon and nitrogen

• The T-DNA region is flanked by 25 bp sequences which is essential for its transfer

• Vir region is also essential for transfer of T-DNA

vir region

• The vir region (35 kbp) is organized into 6 operons

• vir A, vir B, vir C, vir D , vir E and vir G• Genes A,B,D and G essential for virulence• vir C and E are required for tumor induction• Vir A sense the phenolic compounds like

acetosyringone and hydroxyacetosyringone

Vectors based on Ti plasmids &Ri plasmids

• The following properties of Ti plasmids did not allow them to be used directly as vectors

Large size(200-800 kb)-usually small version is preferred

Absence of unique restriction enzyme sites

Tumor induction property

Auxin and cytokinin genes should be removed as it leads to production of these hormones in plants which can prevent or hinder plant growth

Opine synthesis gene as it is not useful to transgenic plant and it also lowers yield

• Recombinant DNA technology has overcome these constraints and a number of Ti plasmid vectors have been created

• Two plasmid derived cloning vector systems1.The binary cloning vector2.The co integrate cloning vector

The Binary vector system contains

• Origin of replication for both E.coli &A.tumefaciens

• A selectable marker gene – to check the transformation efficiency

• A target gene – both of these inserted between T-DNA left and right border

A binary T-DNA plasmid for delivering the Bt gene to plants (not a cointegrate vector)

(NPT or kanr)(35S-Bt gene-tNOS)

(Spcr)

Transfer the Promoter into the T-DNA Shuttle Vector

Use Shuttle vector and Helper Ti plasmid instead of Ti plasmid

The Cointegrate vector contains

• Origin of replication for E.coli• A T-DNA right border• A plant selectable marker gene• A target gene• A sequence of Ti plasmid DNA that is

homologous to a segment on the disarmed Ti plasmid

The disarmed Ti plasmid contains

• The T-DNA left border• The vir gene cluster• An A.tumefaciens origin of replication (ori)

• The recombination between the cointegrate vector and disarmed Ti plasmid results in the final recombinant Ti plasmid

Strategies to avoid Bt resistant insects

• Use of inducible promoters (that can be turned on only when there is an insect problem)

• Construction of hybrid Bt toxins• Introduction of the Bt gene in combination with

another insecticidal gene• Spraying low levels of insecticide on Bt plants • Use of spatial refuge strategies