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Emmer wheat LRR-receptor protein kinase gene, and cloning method and use thereof

A receptor protein, emmer technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as limiting gene function

Inactive Publication Date: 2009-04-15
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ancestral gene of the TaXa site may encode a polypeptide consisting of 1028 amino acids, but so far, there has been no report of a complete, full-length gene cDNA clone encoding about 1028 amino acids isolated from common wheat, which limits the understanding of this Further Study on Gene Function of Homologous Loci

Method used

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  • Emmer wheat LRR-receptor protein kinase gene, and cloning method and use thereof
  • Emmer wheat LRR-receptor protein kinase gene, and cloning method and use thereof
  • Emmer wheat LRR-receptor protein kinase gene, and cloning method and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The cloning process of embodiment 1.TtLRR-STK gene:

[0038] The TtLRR-STK gene was isolated from emmer wheat, which is highly resistant to wheat diseases. Extract total RNA (ribonucleic acid) from the leaves of emmer wheat, further isolate mRNA (messenger ribonucleic acid), transcribe mRNA into cDNA (complementary deoxyribonucleotides) by reverse transcription, and use the cDNA as a template for Gene cloning.

[0039] A pair of degenerate primers (sequence: 5'-GTATTG(A / G)TTTTCTTTGCCTGG(A / C)C-3'; 5'- CAAGC(A / C)GT(A / C)CGTCCAATC(T / A)AT-3'), a cDNA fragment with a coding sequence length of 3081bp was obtained from emmer wheat cDNA by reverse transcription PCR (RT-PCR) technology , cloned on the pUC18-T vector, sequence analysis after sequencing proved that this cDNA clone encodes leucine-rich region-serine / threonine receptor protein kinase (LRR-Serine / Threonine receptor protein kinase). The similarity query and comparison in GenBank proved to be a new emmer gene, tentat...

Embodiment 2

[0049] Example 2. Application of the TtLRR-STK gene of the present invention in transgenic disease resistance breeding.

[0050] The TtLRR-STK gene was digested from the pUC18-T vector, or amplified with high-fidelity Taq-DNA polymerase and gene-specific primers. Use T4-DNA ligase to connect TtLRR-STK to the downstream of maize ubiquitin high-efficiency promoter Ubi (Ubiquitin-1), and then insert it into the pCAMBIA-Bar plant expression vector to construct the pCAMBIA-Ubi-TtLRR-STK expression vector plasmid. The expression vector is transformed into Escherichia coli DH10B bacterial strain for propagation, and the plasmid of the vector is extracted to carry out transgenic disease-resistant breeding. Transform the pCAMBIA-Ubi-TtLRR-STK expression vector plasmid into the callus of immature embryos of wheat varieties with high yield and high quality but poor disease resistance by gene gun (such as Bio-Rad’s high-pressure helium gene gun, PDS-1000 / He) middle. Using the selection...

Embodiment 3

[0051] Example 3. The TtLRR-STK gene of the present invention is designing and synthesizing new disease resistance-related genes.

[0052] Modern DNA synthesis technology has been able to synthesize full-length genes. Therefore, according to the characteristics of the plant LRR-protein kinase disease-resistant genes that have been cloned and studied, the sequence encoding the specific LRR domain required for synthesis is designed and synthesized, and it can be synthesized by linking with the sequence encoding about the 600th amino acid of TtLRR-STK Novel LRR-serine / threonine receptor protein kinase gene. Then, the correct coding frame (ORF) of the synthetic gene was verified by sequencing. The verified gene can be further constructed into a plant expression vector, and the disease resistance of the synthetic gene can be tested by transgenic technology. Synthetic genes with good disease resistance can be used in the breeding of new transgenic disease-resistant wheat varieties...

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Abstract

The invention relates to the technical field of plant gene engineering, in particular to the separation, cloning and applications of a TtLRR-STK gene the full length cDNA of which is 3081bp. The TtLRR-STK gene is separated from the emmer with high resistance to wheat diseases; the gene codes the receptor protein kinase of a leucine-rich area-serine / threonine; and the receptor protein kinase includes a plurality of parts such as an N-end conserved sequence, a leucine-rich structure domain (LRR), a middle transmembrane structure domain, a C-end protein kinase structure, and the like. The structure thereof is similar to a cloned rice resistance to bacterial blight protein Xa21 and is highly alike to one coding protein of the receptor kinase. The TtLRR-STK gene can be inserted into the downstream of promoters with different types for building a plant expression vector and carrying out plant disease resistance improvement through the gene engineering technology, and moreover, the cloning of the gene can also be used for manufacturing the gene chip of the wheat and be applied to the researches of wheat disease resistance, and the like.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the separation, cloning and application of a full-length cDNA. Background technique [0002] The protein kinase family is a large family of enzymes that participate in signal transduction pathways and play an important role in regulating cell functions, many of which mediate the response of eukaryotic cells to external stimuli. Receptor-like protein kinases (RLKs) belong to a subfamily of protein kinases, and their structure includes an extracellular domain (extracellular domain), a single-pass transmembrane domain (signle-pass transmembrane domain) and an intracellular kinase domain ( cytoplasmicprotein kinase domain), they activate the autophosphorylation and mutual phosphorylation activities of the intracellular kinase domain through the specific combination of the extracellular domain and extracellular signal molecules, such as ions, small molecul...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/82
Inventor 牛吉山刘瑞郑磊
Owner HENAN AGRICULTURAL UNIVERSITY
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