Use of r-genes as a selection marker in plant transformation and use of cisgenes in plant transformation

A kind of technology of gene and plant, applied in the field of plant transformation of Solanaceae plants preferably potato

Inactive Publication Date: 2010-03-03
COOP AVEBE U A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a need for alternative transformation methods

Method used

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  • Use of r-genes as a selection marker in plant transformation and use of cisgenes in plant transformation
  • Use of r-genes as a selection marker in plant transformation and use of cisgenes in plant transformation
  • Use of r-genes as a selection marker in plant transformation and use of cisgenes in plant transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Selection of homologous gene R3a transformants

[0121] Construction of vector pBINmf::R3a

[0122] The binary vector pPGB-1S (Kuipers et al., 1995) derived from pBI121 was digested with the enzymes PmeI and ClaI to remove the NptII gene. ClaI sticky ends were blunt-ended by Klenow polymerase treatment, followed by circularization of the vector DNA by blunt-end ligation using T4 DNA ligase. This generated the vector pPGBmf (without marker) containing the potato GB SSI promoter followed by the NOS terminator (as a 1140-bp HindIII / EcoRI fragment) in the T-DNA. The construct pPGBmf was digested with HindIII and EcoRI to obtain two fragments of 1140 bp and 9681 bp. A 9681-bp fragment containing the internal LB (left border) sequence, LB, backbone vector DNA, RB and internal RB sequence was separated from the agarose gel.

[0123] Double-stranded oligonucleotides were prepared by annealing primers AWO1 (5'-AGCTTGGCGCGCCCGGGTTAATTAAG-3') and AWO2 (5'-AATTCTTAATTAACCCGGGCGC...

Embodiment 2

[0144] Selection of homologous gene R3a transformants

[0145] Construction of vector pBINAW2b::R3a with reduced T-DNA borders

[0146] A new backbone vector comprising RB and LB was constructed using pBIN19 as a starting material. Primers for digesting RB and LB. The RB upstream DNA sequence was amplified from 1156 bp pBIN19 using primers URB (5'-GCGGTCCTGATCAATCGTCAC-3') and RBK (5'-GGTACCTGACAGGATATATTGGCGGGTAAA-3'; with KpnI site). The LB downstream DNA sequence was amplified from 627 bp pBIN19 using primers LBKX (5'-GGTACCTCTAGAGTTTACACCCAATATCC-3'; with KpnI and XbaI sites) and DLB (5'-GCGGGTTTAACCTACTTCCTTT-3'). Both PCR products were cloned into pGEM-T and sequenced. Digestion with SacI and KpnI liberated the RB upstream sequence from the pGEM-T vector. LB downstream sequences were released from the pGEM-T vector by digestion with KpnI and NsiI. Fragments were separated from the agarose gel. The SacI / KpnIRB upstream sequence and the KpnI / NsiI LB downstream sequen...

Embodiment 3

[0182] GBSS-IR construct without tag

[0183] Construction of All Potato GBSS-IR T-DNA Vectors

[0184] The T-DNA sequence was entirely derived from the potato GBSSI genome sequence. For the cloning of the extended GBSSI promoter, the HindIII site upstream of commonly used promoters was determined (Visser et al., 1991b; van der Leij et al., 1991a; accession number X58453) up to 0.6 kb upstream of the BglII site (van der Leij et al., 1991a) the sequence of. This PCR was performed using the DNA of plasmid pWAM101 (van der Leij et al., 1991b) as a template with primers GBSS-0 (5'-TACCGCTACCACTTGACATTC-3') and BINMCS (5'-GCACCCCAGGCTTTACACTTT-3'). The 738-bp sequence between BglII and HindIII is highly homologous (98% identity) to the sequence published by Dai et al. (1996) (Accession No. X83220). Primers were designed to amplify the GBSSI promoter and upstream region: primer UPGBX (5'-CTCTAGAAGTTCGAGACACTGGCTACG-3'; with XbaI site) and primer PGBB (5'-GGATCCTGGAGGAGATGAGTAAAAG...

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Abstract

The invention relates to the field of plant transformation, in particular plant transformation of a Solanaceae, preferably of potato. The present invention provides an alternative selection method inplant transformation processes. The invention further provides a plant that has been provided with additional nucleic acid sequences but which genetically modified plant essentially consists of cis plant sequences, for example a genetically modified potato plant that has been provided with additional (essentially) potato plant sequences. Such a transgenic plant is free from non-Solanum T-DNA border sequences. A preferred embodiment is a potato plant that carries a functional R-gene, providing resistance against an oomycete pathogen, preferably Phytophthora infestans, wherein said R-gene can beused as selectable marker.

Description

technical field [0001] The present invention relates to the field of plant transformation, in particular the plant transformation of Solanaceae plants, preferably potatoes. Background technique [0002] Since the discovery in the late 1970s and early 1980s that the Ti plasmid (tumor-inducing plasmid) of Agrobacterium spp. can be used as a carrier in plant genetic engineering, it has been known to use transformed bacteria such as Agrobacterium for plant growth. Transformation to obtain transgenic plants expressing a heterologous gene or gene fragment of interest. The wild-type plasmid induces tumor cells in plant cells, but it can be modified so that it can carry a foreign gene construct into the cell without making the recipient cell tumorigenic. During tumor induction, a specific segment of the Ti plasmid called T-DNA (transfer DNA) integrates into the host plant nuclear DNA. In the genetic engineering of plants, the T-DNA is modified and carries the exogenous gene constr...

Claims

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Application Information

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IPC IPC(8): A01H5/00C12N15/82
CPCC12N15/821C12N15/8282A61P43/00
Inventor 尼古拉斯·克莱门斯·玛丽亚·亨里克斯·德·韦滕理查德·杰勒德斯·弗朗西斯库·维瑟埃弗特·亚科布森埃德温·安德列什·杰勒德·范·德·福森安娜·玛丽亚·艾格尼丝·沃尔特斯
Owner COOP AVEBE U A
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