Novel effective conversion method of helianthus and oil plant seed based on positive selection

A sunflower and seed technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of antibiotic resistance genes, death of transformed cells, cutting off the nutrient supply of transformed cells, etc.

Inactive Publication Date: 2008-12-31
阿维沙基因有限公司
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  • Abstract
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Problems solved by technology

The most obvious disadvantage of these is that non-transformed cells are killed in the presence of phytotoxic products and in cases where connective tissue is used there is a risk that transformed cells also die, since the death of non-transformed cells may cut off nutrition to transformed cells supply or because damaged or dying non-transformed cells may secrete toxic compounds
In addition, the presence of antibiotic resistance genes in ingested plants is worrying

Method used

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  • Novel effective conversion method of helianthus and oil plant seed based on positive selection
  • Novel effective conversion method of helianthus and oil plant seed based on positive selection
  • Novel effective conversion method of helianthus and oil plant seed based on positive selection

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Experimental program
Comparison scheme
Effect test

Embodiment

[0043] Example: 1 Clone xylose isomerase from Thraustochytrid strain SC-1.

[0044] Total RNA was isolated from a three day old culture of Schizochytrium SC-1 (Thraustochytrid isolated from stagnant water in Goa). cDNA was synthesized from mRNA using Superscript RNase (GibcoBRL). The cDNA was cloned into the NotI-SalI sites of the pSPORT1 vector and transformed into E. coli DH10B.

[0045] The primary cDNA library consisted of 2 × 10 6 clones constituted, and the amplified library had a titer of 2×10 10 clones / ml. EST clones were randomly picked and the insert was amplified with SP6 and T7 primers. The 5' ends of 2000 cDNA clones were randomly selected and sequenced with T7 primers. 5' end sequencing of clones from the cDNA library of SC-1 led to the identification of a 1.5 kb cDNA clone encoding a 1511 bp full-length xylose isomerase (xylA) cDNA with a 158 bp 5' UTR and a 30 bp 3' UTR. This clone has an ORF of 1481 bp.

[0046] The sequence of the CDS is shown in SEQ I...

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Abstract

The invention describes a highly improved, reproducible and a consistent method of transformation and regeneration that results in obtaining 12-15% transgenic plants. The present invention relates to a method of selecting genetically transformed Sunflower explants based on their ability to utilize Xylose as a sole carbohydrate source. Further disclosed is the nucleic acid sequence of the Xylose Isomerase gene, vector construction for incorporation of the selection marker gene and the process of Agrobacterium Mediated Transformation of target host plant with the vector comprising the gene encoding the enzyme Xylose isomerase under the functional combination of the heterologous regulatory sequences. Also disclosed is the method of selecting the putative transformants post transformation with the said vector that possess a metabolic advantage of utilizing Xylose as a sole carbon source.; Increased efficiency of regeneration, better growth and survival has been observed on subjection to the described method of positive selection. The subject invention alleviates the disadvantages of negative selection methods such as the undesired elimination of the transformed cells and the potential environmental harm caused due to the dispersal of the antibiotic and the herbicide resistant genes.

Description

Field of invention: [0001] The sunflower (Helian thus annuus) or sunflower is a recalcitrant species that is extremely difficult to transform. We report a highly improved transformation method which leads to a 2-3 fold increase in the number of transformants obtained compared to the cited prior art. The present invention relates to a method of selecting genetically transformed sunflower explants based on their ability to utilize xylose as a sole sugar source. The present invention also discloses the nucleic acid sequence of the xylose isomerase gene, the vector construct for incorporating the selectable marker gene, and the use of vectors comprising the gene encoding xylose isomerase under the control of heterologous regulatory sequences to target hosts. A method for Agrobacterium-mediated transformation of plants. The present invention also discloses a method of selecting putative transformants having a metabolic advantage of utilizing xylose as a sole carbon source after t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/92C12N15/61
CPCC12N9/92C12N15/821
Inventor P·V·莫拉瓦拉K·R·拉伊亚什里
Owner 阿维沙基因有限公司
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