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Corn disease-resistance related gene MR4, and application of the same in corn disease-resistance improvement

A disease-resistant gene, corn technology, applied in plant gene improvement, application, genetic engineering, etc., can solve the problems of slow progress in disease-resistant gene research and weak disease-resistant genetic engineering

Inactive Publication Date: 2013-10-16
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Npr1 is a transcription factor involved in the defense response of Arabidopsis and other plants. Overexpression of Npr1 can improve the disease resistance of Arabidopsis and other plants (Cao et al.1998); Pto (Tang et al.1999), Cf-9 (Hammond et al.1998), Xa21 (Wang et al., 1996), N (Witham et al.1996) and other disease resistance genes have also been cloned and used in crop resistance genetic engineering, but originated from Research on disease resistance genes in maize is progressing slowly, and disease resistance genetic engineering is relatively weak

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  • Corn disease-resistance related gene MR4, and application of the same in corn disease-resistance improvement
  • Corn disease-resistance related gene MR4, and application of the same in corn disease-resistance improvement
  • Corn disease-resistance related gene MR4, and application of the same in corn disease-resistance improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Cloning of maize disease resistance gene MR4

[0020] The previous research in our laboratory found that the expression profiles of maize before and after inoculation with Leptosphaeria spp. ID NO: 1), after comparison and analysis in the NBCI gene database BLAST, it is a similar disease resistance gene, named eMR4. Compared with the existing maize complete gene sequence and expression tag (EST) public database (http: / / www.maizegdb.org / ; http: / / www.maizesequence.org / index.html), we finally found the sequence containing EST The full-length cDNA sequence of the MR4 gene. The nucleotide length of the open reading frame (ORF) of the MR4 gene is 2727bp, which encodes a similar disease resistance protein consisting of 908 amino acids. The encoded protein contains the unique functions of disease resistance genes such as LRR, NB-ARC and AAA-ATPase domain (such as figure 1 shown).

[0021] A pair of primers fulMR4-F (5′-ATGGAATTCGTGGCGTCCAT-3′) that can amplify th...

Embodiment 2

[0023] Example 2: Construction of corn disease resistance gene MR4 overexpression vector and transformation of corn

[0024] The vector used in the present invention is a genetic transformation vector transformed on the basis of the universal binary vector pTF102. Firstly, the multiple cloning site of the original universal binary vector pTF102 was excised, and then the 2×35S promoter upstream of GUS was replaced with the maize ubiquitin gene (Ubiquitin) promoter to obtain a genetic transformation vector named pTE102-M (such as figure 2 shown). After maize transformation, the gene driven by the maize ubiquitin promoter can be expressed stably and efficiently. The maize disease resistance gene MR4 cloned in Example 1 was digested with restriction endonucleases BamHI and KpnI, and the DNA fragment containing the complete reading frame of maize disease resistance gene MR4 was recovered, and the DNA fragment was connected to pTF102 digested with the same enzymes -M vector (the ...

Embodiment 3

[0026] Example 3: Maize genetic transformation and identification mediated by Agrobacterium

[0027] The method for cultivating disease-resistant corn by expressing the MR4 gene is as follows:

[0028] The conventionally planted corn inbred line (named H99) was bagged and isolated when the flowering stage was before ear silking, and self-pollination was assisted artificially. Continue bagging and isolation after pollination. From the first pollination, pollinate again at 24 hours and 48 hours respectively. Remove the whole ear of corn and put it in 75% alcohol for 5 minutes. After drying, the intact immature embryos were peeled off with a sterile blade, further sterilized with 75% alcohol and 0.1% mercury liter, washed with sterile water and dried. The maize disease resistance gene MR4 expression vector pTF102-MR obtained in Example 2 was introduced into the dried immature embryos by the method mediated by Agrobacterium. Transformed immature embryos were co-cultured in the d...

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Abstract

The present invention relates to a corn disease-resistance gene MR4, protein encoded by the corn disease-resistance gene MR4, an expression vector and an application of the corn disease-resistance gene MR4, wherein the nucleotide sequence of the corn disease-resistance gene MR4 is represented by the sequence table SEQ ID NO:1, and the amino acid sequence of the protein encoded by the corn disease-resistance gene MR4 is represented by SEQ ID NO:2. The expression vector construction method comprises: cutting multiple cloning sites of a universal binary vector pTF102, replacing 2*35 S promoter on the upstream of GUS gene by using corn ubiquitin gene promoter to obtain a genetic transformation vector; adopting restriction enzyme BamHI and KpnI to digest the corn disease-resistance gene MR4, and recovering the DNA fragment; and inserting the DNA fragment into the restriction enzyme digestion site of the genetic transformation vector. According to the present invention, the corn disease-resistance gene MR4 is transformed into disease sensitive corn so as to obtain the transgene corn with corn disease-resistance gene MR4 over-expression, wherein the transgene corn has strong fungal disease resistance ability.

Description

Technical field: [0001] The invention belongs to the technical fields of plant molecular biology and plant genetic engineering, and in particular relates to the cloning of a maize disease resistance-related gene MR4, the construction of a plant expression vector and its application in improving maize disease resistance. Background technique: [0002] Disease is an important reason that restricts the increase and stability of grain production. It is estimated that 30% of the annual grain production reduction is caused by disease. Therefore, disease control is an important way to ensure grain production increase and stability. Traditional disease control strategies are mainly to breed disease-resistant varieties through cross-breeding, or to assist in the application of chemical pesticides and chemical inducers. The genetic engineering of plants has shown great potential in the past 30 years of development, and the use of genetic engineering to carry out molecular breeding for...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 张世宏魏毅刘金亮贾保磊李飞武
Owner JILIN UNIV
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