Transcription factor coming from leymus chinensis and related to low temperature resistance, and coding gene and application thereof

A coding and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as difficulty in plant drought resistance and complex plant stress resistance traits.

Active Publication Date: 2014-02-12
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of plant stress resistance traits, it is very difficult to improve the drought resistance of plants using traditional breeding methods
Genetic engineering methods such as transgenics provide an effective way for plant cold resistance breeding, but the isolation of high-efficiency cold resistance genes is the key factor limiting plant cold resistance genetic engineering

Method used

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  • Transcription factor coming from leymus chinensis and related to low temperature resistance, and coding gene and application thereof
  • Transcription factor coming from leymus chinensis and related to low temperature resistance, and coding gene and application thereof
  • Transcription factor coming from leymus chinensis and related to low temperature resistance, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Obtaining the gene LcWRKY3 related to plant low temperature tolerance

[0073] 1. Plant material processing and total RNA extraction

[0074] The seedlings of Leymus chinensis (Trin.) variety Jisheng No. 1 (Jisheng Leymus chinensis seedling station in Jilin Province) were used as materials, and total RNA was extracted after 6 hours and 12 hours treatment at 4°C, and detected by 1% agarose gel electrophoresis. , The result is figure 1 Shown. The extracted RNA has two obvious electrophoresis bands, 28S RNA and 18SRNA from top to bottom, indicating that a more pure and complete total RNA was obtained.

[0075] 2. Obtaining and PCR detection of the full-length cDNA sequence of the gene LcWRKY3 related to plant low temperature tolerance

[0076] The 3'-end sequence of the gene LcWRKY3, which is related to plant low temperature tolerance, was obtained by sequencing in our laboratory. BLAST alignment was performed on the NCBI with the obtained 3'-end cDNA sequence of the ...

Embodiment 2

[0080] Example 2. Bioinformatics analysis of LcWRKY3 and its encoded protein

[0081] 1. Sequence analysis of LcWRKY3 gene and prediction of structure and function of the encoded protein

[0082] Using DNAMAN and DNAstar software, the full-length cDNA sequence of LcWRKY3 obtained in Example 1 was analyzed by bioinformatics, and the protein LcWRKY3 was encoded by 338 amino acid residues. It is estimated that its molecular weight is 35.511kDa, and its isoelectric point PI value is 9.752. The online Blast tool (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to analyze the domain of LcWRKY3, and the results showed that the sequence of LcWRKY3 between amino acid residues 270-330 is a conservative WRKY DNA binding domain, the sequence between amino acid residues 221-227 is a possible nuclear localization signal PRRRCSG. This indicates that the protein is a member of the WRKY transcription factor superfamily.

[0083] 2. The homology and phylogenetic tree analysis of the amino acid se...

Embodiment 3

[0085] Example 3. Analysis of expression pattern and tissue specific expression of LcWRKY3 under different abiotic stress and hormone treatment conditions

[0086] 1. The expression pattern analysis of LcWRKY3 under different abiotic stress and hormone treatment conditions

[0087] The seedlings of Leymus chinensis (Jisheng No.1, Jisheng Leymus chinensis Fine Breeding Station, Jilin Province) which had been grown normally for 9 weeks were subjected to different abiotic stress: 400mM NaCl, 42℃ high temperature, 4℃ for 6 hours and different hormones: 100μM 6-benzyl adenine (BAP), 100μM salicylic acid (SA), 100μM methyl jasmonate (MJ), 100μM gibberellin (GA), 100μM abscisic acid (ABA) for 6 hours. The total RNA of Leymus chinensis seedlings was extracted separately, using LcWRKY3 gene primers (upstream primer: 5'-AAGCTGGTCCAGCCATTGTC-3' (sequence 2 370-389); downstream primer: 5'-CGTCCTCTTCACCCGCAAC-3' (sequence 2 reverse complementary sequence of positions 750-768)) with Actin gene ...

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Abstract

The invention discloses a transcription factor coming from leymus chinensis and related to low temperature resistance, and a coding gene and application thereof. Protein provided by the invention is shown as the following (a) or (b): (a) protein composed of an amino acid sequence shown as sequence 1 in a sequence table; and (b) protein derived from the protein of the sequence 1, wherein the protein of the sequence 1 is subjected to substitution and / or deletion and / or adding of one or multiple amino acid residues which are related to plant cold resistance. Experiments prove that: after subjected to stress treatment at a low temperature of -8 DEG C for 24 h, at the 14th day, about 81% of wild-type arabidopis thaliana plants have the symptoms that leaves have water loss and are wilted, the color of leaves is whitened, and the plants are partially or completely dead, while about 84% of trans-LcWRKY3-genic arabidopis thaliana sprout plants grow normally, only about 16% of the plants have the symptoms that leaves are whitened; and therefore the transcription factor provided by the invention has important theoretic and practical meanings on cultivation of plant novel species with improved cold resistance, is applicable to cultivation and identification of resistance plant species required by husbandry and ecological environment treatment, and has relatively high practical application value.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and relates to a protein derived from Leymus chinensis and related to plant low temperature tolerance and its coding genes and applications, in particular to a Leymus chinensis cold-induced transcription factor derived from WRKY family and its coding genes and applications Application in cultivating plants with improved low temperature tolerance. Background technique [0002] Low temperature stress is one of the main obstacles affecting the normal growth of plants. The cold resistance of plants, especially cash crops, directly affects crop yields. At present, the identification of cold-tolerant genes is mostly concentrated in model plants such as Arabidopsis and rice, and important crops such as corn and cotton. Most of them are plants of tropical origin, and they cannot tolerate the cold environment, causing damage at 0℃~12℃. Therefore, it is urgent to dig out related genes from other plants wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/21C12N1/19A01H1/02C12N15/82C12N15/84A01H5/00
CPCC07K14/415C12N15/8273
Inventor 刘公社程丽琴赵爱国陈双燕齐冬梅
Owner INST OF BOTANY CHINESE ACAD OF SCI
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