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Cold-induced COR15a gene of capsella bursa-pastoris and application thereof to cold resistance of improved plants

A cold-induced, shepherd's purse technology, applied in the field of plant genetic engineering, can solve problems such as cold-resistant plants that have not yet been found

Inactive Publication Date: 2011-02-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a variety of cold-regulated proteins have been found in plants, but the amino acid composition, sequence, repeat unit, repeat number, spatial structure, and tissue-specific distribution of different cold-regulated proteins vary among different plants and within the same plant. Show a certain degree of diversity, and their composition and structure also show diversity and complexity
So far, no cold-regulated gene has been found CbCOR15a and related reports on the use of this gene to breed cold-resistant plants

Method used

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  • Cold-induced COR15a gene of capsella bursa-pastoris and application thereof to cold resistance of improved plants
  • Cold-induced COR15a gene of capsella bursa-pastoris and application thereof to cold resistance of improved plants
  • Cold-induced COR15a gene of capsella bursa-pastoris and application thereof to cold resistance of improved plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Isolation and Identification of Shepherd's Purse COR15a Gene

[0075] 1. Cultivation of shepherd's purse seedlings

[0076] The shepherd's purse seeds come from Shanghai Seed Company. After the shepherd's purse seeds are sterilized, they are sown in the 0 The culture tank of the culture medium was placed at 25° C. for 4 weeks, and the photoperiod was 16 hours of light and 8 hours of darkness.

[0077] 2. DNA isolation

[0078] Take shepherd's purse leaves, add liquid nitrogen, grind into powder, and use CTAB method to extract DNA. The quality of DNA was identified by agarose gel electrophoresis, and then the content of DNA was determined on a spectrophotometer.

[0079] 3. Cloning of genes

[0080] According to Arabidopsis ( A. thaliana ),rape( Brassica napus ) and shepherd's purse ( C. bursa - pastoris ) in the amino acid coding region of the COR gene (GenBank Accession No.: AY437888), design a pair of primers (respectively denoted as SEQ ID N...

Embodiment 2

[0088] Example 2 CbCOR15a Bioinformatic analysis of genes

[0089] 1. Homology analysis

[0090] CbCOR15a and other members of the COR15 protein family include Arabis pumila ApCOR15a (AAS98217) in A. thaliana AtCOR15a and AtCOR15b in (AAD22999 and AAD23000), C .bursa-pastoris CbCOR15b (AAR99417) in Arabidopsis lyrata subsp. lyrata AlCOR in (XP_002881858), Eutrema salsugineum EsCOR (ABX79675) in Draba alpina DaCOR15a (ABW71518) in, D. draboides DdCOR15a (ABS50438) in, B. napus BnBN115 (AAA66068) in, Chorispora bungeana CbCOR15 (ABM97656) in B. rapaThe amino acid sequence of BrCOR (ABF60663) was analyzed by Protein-Protein BLAST (http: / / www.ncbi.nlm.nih.gov / blast) and Vector NTI v10.0 software, showing high homology, Among them, ApCOR15a (identity 91%, similarity 94%), AtCOR15a (identity 89%, similarity 91%) and CbCOR15b (identity 80%, similarity 87%) have the highest homology ( figure 1 ).

[0091] 2. Phylogenetic tree analysis

[0092] Using Clustal...

Embodiment 3

[0095] Example 3 Under various induction conditions CbCOR15a Tissue-specific expression analysis of

[0096] Analysis of leaves and roots of shepherd's purse plants after cold acclimation, cold induction and signal molecule treatment CbCOR15a expressive properties. Cold acclimation treatment: 4-week-old shepherd’s purse seedlings grown under normal light conditions at 26°C were treated sequentially at 12°C for 4 days, 4°C for 4 days, 0°C for 2 hours, and -4°C for 2 hours; cold induction treatment: grown at 26°C under normal conditions 4-week-old shepherd's purse seedlings under light conditions were moved to 4°C for 0, 1, 3, 6, 24h; signal molecule treatment: 4-day-old shepherd's purse seedlings grown under normal light conditions at 26°C were treated with 100 μM (±)-cis , trans-ABA, 5μM (±) GA 3 , 500μM (±) SA, 300μM (±) MeJA and 20μM (±) IAA aqueous solution were sprayed for 0h, 1h, 6h, 24h. The total RNA of the three tissues of the lower leaves and rhizomes of each tr...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular discloses a cold-induced CbCOR15a gene of capsella bursa-pastoris and application thereof to cold resistance of improved plants. The nucleotide sequence of the gene is shown as SEQ ID No. 1; the CbCOR15a gene is a nucleic acid sequence which has the 1st to the 674th basic groups in a sequence table SEQ ID No. 1; and a gene which is induced to express at a low temperature over zero, stops or reduces expression at normal temperature and can promote a cell to produce cold resistance after expression is encoded by the nucleic acid sequence. An inductive expression vector constructed by gene segments can express when the plants are stressed by low temperature. The invention also provides an application method for breeding cold-resistant plants based on the gene; and the method is used for improving the varieties of crops.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a shepherd's purse cold-induced gene COR15a ( CbCOR15a ) and a preparation method thereof, and also relates to a recombinant plasmid of the gene, which is applied to plant stress genes, especially for genetic transformation of plant cold resistance genes, so as to achieve the purpose of improving plant cold resistance. Background technique [0002] Plants have sufficient adaptability and resistance to environmental changes and adverse environments. This stress resistance is not only controlled by the genetic type of phylogenetic evolution, but also restricted by physiological and ecological factors in individual development. As one of the important environmental factors, temperature limits the distribution, growth and yield of plants, and plays a decisive role in plant growth and development. Low temperature damage is not only a serious natural disaster in agri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C07K14/415A01H5/00C12N15/10C12N15/70C12N15/82C12N15/29
Inventor 林娟周明琦沈忱吴丽华
Owner FUDAN UNIV
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