Wheat disease-resistant gene Lr-L1 and its use

A disease-resistant gene, wheat technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of less research on the isolation, cloning and disease resistance response of wheat disease-resistant genes

Inactive Publication Date: 2005-07-20
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are relatively few studies on the isolation, cloning and

Method used

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  • Wheat disease-resistant gene Lr-L1 and its use

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Cloning process of wheat disease resistance gene Lr-L1:

[0072] Wheat Lr-L1 gene was isolated from a new wheat disease-resistant line "99-2439" with high resistance to powdery mildew and rust. Total RNA (ribonucleic acid) was extracted from leaves 22 hours after powdery mildew induction, and mRNA (messenger ribonucleic acid) was further isolated, and mRNA was transcribed into cDNA (complementary deoxyribonucleic acid) by reverse transcription, and the cDNA was used as a template Perform gene cloning. A degenerate primer (sequence: 5'-GTGAGGCTT(G / A)A(T / C)(G / A)TC(A / G)AA(G / A) T(G / A)GAG(G / A)ATGCG-3'), many products were obtained by rapid amplification of cDNA end technology (5'-RACE), and an amplification product of about 1500bp was recovered. It was cloned on the pGEM-T Eseay vector, and sequence analysis proved that this cDNA fragment was a part of the receptor-like protein kinase gene after sequencing.

[0073] Using a specialized kit (SMART TM RACE cDN...

Embodiment 2

[0145] Embodiment two: Transgenic disease-resistant breeding:

[0146] Digest the Lr-L1 gene from the pGEM-T easy vector, or amplify it with high-fidelity Taq-DNA polymerase and gene-specific primers. Use T4-DNA ligase to connect Lr-L1 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-Lr-L1 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-Lr-L1 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 marker gene on the expression vector—the herbicide-resistant gene Bar, the...

Embodiment 3

[0147] Embodiment three: design and synthesis of new disease resistance gene:

[0148] Modern DNA synthesis technology has been able to synthesize full-length genes. Therefore, according to the difference between the Lr-L1 gene and the LRK10 gene, the cDNA encoding the 1-180 amino acid residues of the LRK10 gene is synthesized and connected with the 176 amino acid residues encoded by the Lr-L1 gene to the end of the gene cDNA, thus synthesizing the Brand new genes. 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 (the construction of plant expression vectors and the specific operations of transgenes are as described in (1)).

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Abstract

The present invention relates to plant gene engineering technology, and is especially one kind of wheat disease resisting gene Lr-L1 and its application. The wheat disease resisting gene Lr-L1 has cDNA of whole length 2326 bp, including 5'-noncoding area of 135 bp, 3'-noncoding area of 249 bp, coding area of 1911 bp, terminator, and polyadenyl acid tail of 28 bp. The 3'-noncoding area includes includes 3 typical tailing signal sequences and can reinforce the tailing process after gene transcription. The wheat disease resisting gene Lr-L1 may be inserted into the downstream of different types of promoter to constitute plant expression vector for plant disease resisting improvement in gene engineering technology, and may be cloned for making wheat gene chip for wheat disease resistance and other research.

Description

[0001] One. Technical field: the present invention relates to the field of plant genetic engineering technology, in particular to a kind of wheat disease resistance gene Lr-L1 and its application. 2. Background technology: [0002] Plant transgenic research originated in the early 1980s. In 1983, Zambryski obtained the first transgenic plant in the world. In 1985, Horch created the leaf disc method in Agrobacterium transformation. Since then, the Agrobacterium-mediated method, virus Mediated method, PEG-mediated method, electroporation method, microinjection method, pollen tube channeling method, ultrasonic method, gene gun method, etc., the species successfully transgenic are constantly expanding, involving more than 50 species and more than 110 kinds of plants, of which , the transgenic plants obtained by the Agrobacterium-mediated method accounted for more than 85% of the total number of transgenic plants, and the transgenic plants obtained by the biolistic method accounted ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29
Inventor 牛吉山郭天财张丽娜
Owner HENAN AGRICULTURAL UNIVERSITY
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