Essay: Exploring pCAMBIA Vectors For Plant Biotechnology Assignments



PLANT BIOTECHNOLOGY ASSIGNMENT

pCAMBIA VECTORS

  COMPILED BY

   RAKSHANAA. R

  4011427

    BMS-BIOTECH

INTRODUCTION ON pCAMBIA VECTORS:

• pCAMBIA VECTORS are Agrobacterium based plasmid vector.

• pCAMBIA VECTORS are the improved version of vectors for PLANT TRANSFORMATION.

• It has a wide range of vectors. Mostly, it is a Kanamycin based binary vectors with plant selection gene and small replicon that can express in plant system.

•  pCAMBIA VECTORS is derived from pPZP vector. This pPZP vector is the Agrobacterium based binary vectors that are very small and versatile. This vector was established by Svab, Maliga & Hajdukiewicz.

• pCAMBIA Vectors was developed mainly for the plant molecular biology community as a resource. Under the Material Transfer Agreement these vectors should be used only for academic or non-profit research organization. If the researchers want to commercially use, a separate license is required. Many configurations of E.coli gusA  gene have been constructed and covered by patents. These patents are owned by CAMBIA.

PLANT TRANSFORMATION:

  Scientific method of inserting foreign DNA into the genome of a plant species of interest is called Plant Transformation. The inserted gene is called Transgene and the resulting plant is called Transgenic Plant. Inorder to facilitate the generation of transgenic plants, there are specially designed plant transformation vectors called the plasmids. The commonly used plant transformation vectors are the binary vectors.

BINARY VECTORS are also called as Shuttle Vectors. They are able to replicate in E.coli as well as Agrobacterium tumefaciens (i.e) in multiple hosts.

PLANT TRANSFORMATION VECTORS have following features:

• For creating a circular strand of DNA Plasmid selection is essential.

• Inorder to work easily Plasmid replication fork needed

• For inserting DNA into agrobacteria T-DNA region is required.

T-DNA also called as Transfer DNA of Tumor inducing plasmid. These plasmids are mostly bacteria like Agrobacterium tumefaciens, Agrobacterium rhizogenes. The length of T-DNA is 24,000 base pairs. At the end of the T-DNA there are 25 base pair repeats and the transfer of T-DNA into the genome of host plants nuclear DNA is initiated at the right border and terminated at the left. They require vir genes of the Tumor inducing plasmid. They have a gene that encodes enzymes for synthesizing phytohormones and opines. The plant hormones synthesized allows the plant cell to grow unconditionally by forming crown gall tumors by Agrobacterium infection. The bacterium uses opines as a source of carbon and energy.

T-DNA BINARY SYSTEM:

   In order to produce a genetically modified plants a pair of plasmids are used. These plasmids are artificial vectors obtained from naturally occurring Ti plasmid.

 Binary Plasmids – Contain transgene, plant selectable marker, bacterial selectable marker and origin of replication site.

 Helper plasmids – Contain vir genes. These genes are from Ti plasmid and they facilitate transduction of T-DNA to host plant cell. They also contain bacterial selectable marker and origin of replication site.

PLANT TRANSFORMATION METHODS:

Among these methods bacterial mediated and direct method of transfer of DNA are most commonly used. However the old binary vectors in these methods have the following drawbacks.

 They have low copy number that results in low yield DNA

 Variable loss of plasmids during propagation because of unstable replicons and large size.

 For bacteria and plant the choice of selectable markers are limited.

 These vectors lack methods to construct reporter gene fusions and convenient restriction sites for manipulation.

In order to overcome these drawbacks alternative vectors were found. One such technically improved vector is the pCambia vector.

FEATURES OF pCAMBIA VECTORS:

1. In E.coli the pCambia vectors have a high copy number which gives very high DNA yield.

2. The vector size depends on which plasmid it is used. Usually they are small in size with 7-12kb.

3. For high stability in Agrobacterium – pVS1 replicon is used.

4. Bacterial and Plant Selection are mostly with Kanamycin. For bacterial even chloramphenicol and for plant hygromycin B also preferred.

5. These vectors construct translational fusion to gusA reporter genes.

6. For introducing the DNA of interest and for modifying the modular plasmids, polylinkers and restriction sites are designed respectively.

pCAMBIA VECTORS FOR CLONING:

    pCAMBIA VECTORS are suitable for cloning because of the addition of selection gene marker (i.e) Kanamycin and also they have fused reporter genes. These genes helps in coding the sequences for GFP and GUS reporter genes. The fused form appears as (GFP::GUS (or) GUS::GFP). The gene product produced by X-Gluc degradation and it was observed by confocal microscope and by selecting the tissues. Also pCambia vectors are suitable for transformation because of their small size, high copy number and kanamycin selection gene marker.

 

  Reasearcher community selects these pCambia type of vectors mainly because of their availability and usefulness.

    Nowadays there are more wide range of vector series.

    pCAMBIA series also have the same features.

There are suitable criteria for using pCambia vectors on transgenic plants. Earlier used vectors like pBI121 have kanamycin selection towards right border of T-DNA and have low copy number. This low copy number hinders in plasmid isolation and cloning. The presence of pCambia vector series that has kanamycin or hygromycin gene selection towards left border of T-DNA helps in eliminating false positive transgenic plants which means plants are negative for gene of interest and positive for markers.

   pCambia vectors have high copy number in E.coli for easy cloning.

   pCambia vectors are small in size. For inserting the gene of insert there are numerous MCS (Multiple Cloning Site)

MULTIPLE CLONING SITE also called as Polylinker. They have many restriction sites. These sites are unique and are present in a short segment in the DNA.

    

    An Array of  pCambia vectors have been developed. Inorder to select the vectors, the researchers should know the objectives of the project and select accordingly.

    pCambia vectors are free to operate. It is an open source so that everyone can use.

   pCambia vectors have a popular promoter, frequently used plant selectable marker and screenable marker.

 Promoter – 35S

 Plant Selectable Marker – Kanamycin or Hygromycin B

 Screenable Marker – GUS

PLANT SELECTION GENE:

   In pCambia vectors the plant selection genes are initiated by double enhancer version of CaMV35S promoter and terminated by CaMV35S poly A signal. This 35S promoter has a augmenter effect on the expression of other genes. The gene expression of pCambia derivatives should be accounted with a caution.

REPORTER GENE:

   In pCambia vectors the reporter gene outlines a hexa-histidine tag at C terminus. This C terminus permits the purification of immobilized metal affinity chromatography resins. In the place of reporter gene, the gene of interest can be inserted. The sequence occurs between the first NheI site and unique Pm/I site. At BstEII site, it will add neither a tag nor a stop codon.

POLYLINKERS:

   The smaller polylinkers eliminates important conflicts from sites (SphI) or XbaI that has ATG and TAG respectively. Because of this other sites in the vectors are useful. (i.e) SphI site in the right T-DNA border and SacII site in the left T-DNA border. Polylinkers widely used was pUC18 but inorder to ease the choice of cloning enzyme, pUC8 and pUC9 polylinkers are used.

NOMENCLATURE OF pCAMBIA VECTORS:

1st Digit denotes Plant selection

 0 – Absence

 1 – Hygromycin resistance

 2 – Kanamycin

 3 – Phosphinothricin

2nd Digit designates Bacterial selection  

 1 – Streptomycin or Spectinomycin resistance

 2 – Chloramphenicol

 3 –  Kanamycin

 4 –  Strep/Spec and Kanamycin

3rd Digit exhibits Polylinkers

 0 – pUC18

 8 – pUC8

 9 – pUC9

4th Digit denotes Reporter gene

 0 – Absence

 1 – E.coli gusA

 2 – mgfp5

 3 – gusA::mgfp5 fusion

 4 – mgfp5::gusA fusion

 5 – Staphylococcus sp.gusA (GUSPlus)

5th Digit indicates Special features

 1 –   Absence of signal peptide from GUSPlus protein and pCAMBIA1305

 2 – Presence of GRP signal peptide for in planta secretion of GUSPlusTM protein

Lagging Letter

 a/b/c – indicates reading frame

 X – denotes absence of own start codon and vector in reporter gene

 Z – denotes presence of lacZa gene for blue/white screening.

TYPES OF pCAMBIA VECTORS :

  There are different types of pCambia vectors, legacy pCambia plasmids and pCambia GUSPlus plasmids are binary vectors whereas pCambia5105 & 5106 are unitary vectors.

 In the legacy complete kit of pCambia plasmids there are 26 different pCambia plasmids and the vectors present in the kit are spotted dried on paper for shipping and they are readily eluted.

LEGACY pCAMBIA VECTOR KIT

STANDARD GUSPlus VECTOR KIT

TRANSBACTER STRAINS/BINARY VECTOR KIT

TRANSBACTER STRAINS/UNITARY VECTOR KIT

VECTOR MANUAL :

pCAMBIA R-gene bacteria R-gene plants Polylinker Reporter gene Vector family Reading frame or lacZ T-DNA size (bp)

5105 Kan+spec/strep hptII gus plus GIS lacZ 5599

2301 kan nptII pUC18 gusA GIS 5391

2300 kan nptII pUC18 MSV 2512

2201 cmr nptII pUC18 gusA GIS 5391

2200 cmr nptII pUC18 MSV 2512

1391 kan hptII pUC9 gusA FUV 4427

1391Z kan hptII pUC9 gusA PCV lacZ 4984

1391Xc kan hptII pUC9 gusA FUV c

1391Xb kan hptII pUC9 gusA FUV b

1391Xa kan hptII pUC9 gusA FUV a

1390 kan hptII pUC9 MSV 2630

1381 kan hptII pUC8 gusA FUV 4427

1381Z kan hptII pUC8 gusA PCV lacZ 4984

1381Xc kan hptII pUC8 gusA FUV c

1381Xb kan hptII pUC8 gusA FUV b

1381Xa kan hptII pUC8 gusA FUV a

1380 kan hptII pUC8 MSV 2630

1304 kan hptII pUC18 gfp:gusA GFP 6128

1303 kan hptII pUC18 gusA:gfp GFP 6128

1302 kan hptII pUC18 gfp GFP 4316

1301 kan hptII pUC18 gusA GIS 5607

1300 kan hptII pUC18 MSV 2728

1291X cmr hptII pUC9 gusA PCV lacZ 4984

1281Z cmr hptII pUC8 gusA PCV lacZ 4984

1201 cmr hptII pUC18 gusA GIS 5606

1200 cmr hptII pUC18 MSV

2727

0390 kan pUC9 DIY 582

0380 kan pUC8 DIY 582

These vectors can be classified accordingly, like

• Minimal Selection Vectors

• Gus Intron Selection Vectors

• GFP Selection Vectors

• Fuse and Use Vectors

• Promoter Cloner Vectors

pCAMBIA5105:

 This pCAMBIA5105 vector is a part of pCambia Gene Transfer Bacterial Acquired Competence with Kanamycin Selection (GT-BACK) Vectors.

 This is an unitary vector.

 It is amodified version of pCAMBIA1105.1

 This vector has a fragment of Ti plasmid from pTiBo542 that has only virA, B, C, D, E, G, K, J operons.

 This is provided as an open source tool kit as it enables the DNA transfer capability to be moved and stabilized on the bacteria.

 In the TransBacter Technology this new vector has a major role. By conjugation function GT-BACK vector eliminates the plasmid to be conjugated and transmissible to othe host because they lack RK2. This RK2 is derived from oriT. They eliminate quarantine issues and the need for compliance with importation controls on living organisms.

 They have host range of origin of replication from pVS1.

 They allow wide range of bacterial spectrum to explore as gene transfer vectors.

 They also enable choices of benign symbionts as they do not have any stress on plants (physical or genetic stress)

 This type of pCAMBIA vector sets are used literally by 1000 of laboratories worldwide.

pCAMBIA0380 ; pCAMBIA0390:

 These vectors have

 Restriction sites on pUC8(0380) and pUC9 (0390) polylinkers

 Promoters

 Selection Genes

 Reporter Genes

 The functional signals are the stop and start codons, NOSpoly (A) signal and histidine tag that signals between the T-DNA border.

 The standard features are present in these vectors.

 Stable replication in Agrobacterium tumefaciens

 Bacterial Selection Kanamycin

 High copy number in E.coli

 

Minimal Selection Vectors:

  pCAMBIA1200;pCAMBIA1300;pCAMBIA1380;

  pCAMBIA1390;pCAMBIA2200;

  pCAMBIA2300:

 For plant transformation these vectors have minimal heterologous sequence.

 Initially they require promoter and terminator.

 They have 2 plant selection genes

 hptII gene that encodes resistance to hygromycin

 nptii gene that encodes resistance to kanamycin

These genes undergo site directed mutagenesis and they eliminate the disturbing restriction sites.

 These vectors undergo co-transformation method (i.e) by separating the vector containing the isolates, selecting the clones and analyzing the plasmid DNA and if necessary by including the plant species and mixing the cell lines before the plant tissue is transformed.

 This method gives T-DNA with 10-30% of transformed plant.

   

    

Gus Intron Selection Vectors:

 pCAMBIA1201;pCAMBIA1301;pCAMBIA2201;

pCAMBIA2301

 Corresponding to the Minimal selection vectors these have pUC18 polylinkers, lacZa and the bacterial, plant selection gene.

 These vectors have their own functional reporter gene, promoter and terminator.

 These vectors are used to create a fusion of gusA with their own gene of interest. This vector uses a GUS assay for simple and sensitive analysis.

 In order to ensure the expression of the activity of glucuronidase from the cells of eukaryotes inside the coding sequence the vector construct uses E.coli gusA with an intron.

 

 

GFP Selection Vectors:

  pCAMBIA1302; pCAMBIA1303; pCAMBIA1304:

    

 These vectors are similar to GUS but include GFP. GFP is a popular reporter gene.

 Based on pCAMBIA1301,1302,1302 they contain mgfp5 version of green fluroscent protein, translational fusion with gusA,gusA:mgfp5His6 fusion and mgfp5:gusA His6 fusion respectively.

 These gusA are intronless.

 Researchers constructed an alternative egfp gene for mgfp5 as this gene was quite faint.

   

Fuse and Use Vectors:

pCAMBIA1381,pCAMBIA1381Xa,pCAMBIA1381Xb, pCAMBIA1381Xc,pCAMBIA1391,pCAMBIA1391Xa, pCAMBIA1391Xb, pCAMBIA1391Xc ORF Variants:

 These vectors are derived from pCAMBIA1380 and pCAMBIA1390

 They have promoterless, non-intron gusA gene in reading frames and polylinkers on either side.

 These have plant and bacterial selection genes.

 These vectors use gusA for reporter gene expression.

 For the construction purpose of transcription and translation fusion gene to the gusA these vectors are used.

 These have one reading frame and they withhold the initation codon of NcoI site.

 

 

 

 

Promoter Cloner Vector:

   pCAMBIA1281Z;pCAMBIA1291Z;pCAMBIA1381Z;  pCAMBIA1391Z:

 Plasmids in this have Chloramphenicol or Kanamycin

 These vectors have promoterless gusA, catalase intron, truncated lacZa that codes for blue/white screening of clones in polylinkers.

 Strong double enhanced 35S promoter is present. Important interference in the expression of pCAMBIA with hptII gene in T-DNA should be observed and interpreted.

 To avoid 35S interferences co-transformation methods should be followed.

 One such method is that, with these promoter cloner vectors one can clone their promoter of interest. In order to clone, these vectors used should undergo self ligation, modification by double digestion of Smal and Xmnl and also undergo gel purification.

   

All these vectors are the improved version of  vectors for plant transformation and they are mainly used for research and mostly for non-profit purposes only.

Reference:

  www.cambia.org

 www.wikipedia.com

 www.itsbio.co.kr

 www.markergene.com

 www.researchgate.net

 The small versatile pPZP family of Agrobacterium binary vectors for plant transformation, Plant Molecular Biology 25:989-984 – Svab, Maliga & Hajdukiewicz

 Binary vector stratergy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid, Nature 303:179-180 – Hooykaas et al

 Vectors for cloning in plant cells, Meth Enzymol 153:277-292 – Hofte et al