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Methods to obtain drought resistant plants

A plant and plant cell technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, enzymes, etc., can solve problems such as screening methods that have not been proposed

Inactive Publication Date: 2013-11-20
高等研究院和国家职业技术学院研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

From these computational studies, it recommends the use of the trehalase gene to generate genetically modified plants in which trehalose metabolism can be altered in the plant, but does not propose its use as a screening method

Method used

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  • Methods to obtain drought resistant plants
  • Methods to obtain drought resistant plants
  • Methods to obtain drought resistant plants

Examples

Experimental program
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Effect test

example 1

[0085] Example 1. Materials and methods

[0086] a) Nucleic acid manipulation

[0087] plasmid DNA. Isolation procedures, restriction, conjugation and transformation of bacterial plasmid DNA were performed according to standard protocols (Sambrook et al., 1989 (Sambrook et al., 1989)). About carrier TOPO (Invitrogen, Inc.) ligation of PCR products was performed according to the manufacturer's instructions. From small scale preparations by RT-PCR, 3'RACE and Sequencing of the obtained fragments.

[0088] plant DNA. To clone the promoting sequence rd29A of Arabidopsis thaliana, phenol:chloroform extraction of genomic DNA was performed from Arabidopsis plants germinated for 10 days in MS medium with 0.7% agar.

[0089] Plant RNA. For cDNA cloning of the trehalase gene from alfalfa, TRI was used according to the manufacturer's instructions. (Molecular Research Center, Inc.), from alfalfa (M. sativa cv. CUF-101) germinated in vitro on MS medium with 0.8% agar for 7 da...

example 2

[0097] cDNA Cloning of Example 2.MsTRE Medicago Gene

[0098] Since the sequence encoding the trehalase from Medicago sativa (termed MsTRE (Ms: Medicago; TRE: trehalase)) was not reported and in order to obtain a fragment for use in a construct inhibiting this enzyme, it was set out to obtain its The complete sequence of DNA. Therefore used: a protocol based on the amplification (RT) of this gene transcript and its subsequent PCR amplification (RT-PCR).

[0099] a) Internal fragment amplification by RT reverse transcription and PCR (RT-PCR)

[0100] A simple test of RT-PCR using the degenerate oligonucleotides Tre300 and Tre351 for the amplification of internal fragments of the cDNA was based on the Brassica thaliana trehalase (Arabidopsis thaliana; deposit accession number AAF22127), potato (potato; accession number A67882) and soybean (glycine max; accession number AAD22970) in the conserved region. Sequence alignment was performed using the Clustal method (DNA Star Softw...

example 3

[0110] Example 3. Design of antisense TRE sequences (TREas) and interfering RNA (Tre-RNAi)

[0111] a) Amplification and cloning of TRE internal fragments with large homology between species.

[0112] In order to express the antisense sequence of trehalase under the promoters 35S and rd29A in plants, a fragment (SEQ.ID.No.18) containing a 560bp sequence internal fragment was first selected, which includes conserved among different plant trehalases small area. To this end, a pair of specific oligonucleotides Tre333 and TreNsiRw was designed, which amplifies an internal fragment (TRE) selected from the DNA plasmid of the vector TOPO-3Tre. The TreNsiRw oligo introduced an Xma I site followed by an Nsi I site into the 3' end of the sequence. Cloning the amplified product into the vector TOPO TA In , the plasmids TOPO-tre560s and TOPO-tre560as were generated due to the bidirectional insertion of the fragments into the vector. In both constructs, the insert is flanked by the Sa...

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Abstract

The present invention provides a nontoxic method for the selection of transformed cells from a cell population consisting of transformed and untransformed cells. The method comprises the following steps: a) introducing into a cell at least one nucleotide sequence of interest and at least one selection nucleotide sequence to obtain genetically transformed cells, where the selection nucleotide sequence comprises a sequence promoting the inhibition of endogenous trehalase enzyme; b) placing the population of transformed and untransformed cells in a culture medium containing an osmoregulating substance such as PEG 8000; c) selecting the transformed cells from the population based on the ability of transformed cells to survive and grow in the presence of the osmoregulating substance conferring the plants derived from these genetic transformation events, for example, drought and / or cold tolerance capacity.

Description

technical field [0001] The present invention relates to genetic engineering, in particular to methods of transgenesis and cisgenesis of plants and tissues of plant origin, and more particularly to methods of selecting genetically transformed plant cells which avoid the use of resistant genotoxicity The substance acts as a selectable marker and renders the transformed cell free from producing proteins and / or new or different metabolites with potential adverse effects on said plant cell, plant or its end users and intermediates. Thus, the method of the present invention uses a nucleotide sequence comprising a nucleic acid sequence that promotes inhibition of endogenous trehalase, which confers drought tolerance to plants obtained by this selection method. Background technique [0002] It is well known that when genetic material is introduced into a population of cells by transformation, only a certain number of cells are successfully transformed. Following transformation, the...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N9/24
CPCC12N15/821C12N9/2402C12N15/8273C12N15/82
Inventor 肯尼·亚历山大·阿格雷达拉古纳乔斯·路易斯·卡夫雷拉庞塞安娜里·戈麦兹埃斯科韦多路易斯·拉斐尔·埃雷拉埃斯特拉罗伯托·蒙特斯德卡卢纳罗伯托·瑞兹梅德拉诺比阿特丽斯·埃克色科诺斯特卡扎雷斯
Owner 高等研究院和国家职业技术学院研究中心
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