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Novel cotton fungal disease-resistant gene GhMPK7 and application thereof

An antifungal, genetic technology, applied in the field of molecular biology and biology

Inactive Publication Date: 2010-07-21
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] After searching, there is no report about the cotton disease resistance-related gene GhMPK7 in domestic and foreign literatures and patents

Method used

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  • Novel cotton fungal disease-resistant gene GhMPK7 and application thereof
  • Novel cotton fungal disease-resistant gene GhMPK7 and application thereof
  • Novel cotton fungal disease-resistant gene GhMPK7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach ( 1

[0121] Embodiment (1): Cloning of new cotton anti-fungal disease-related gene GhMPK7

[0122] 1. Extraction of RNA: Extraction of cotton total RNA by Trizol method

[0123] 2. Synthesis of the first strand of cDNA

[0124] Follow the instructions of the EasyScript First-Strand cDNA Synthesis SuperMix kit.

[0125] Add the following reagents to a 0.25ml centrifuge tube:

[0126] Total RNA 5.0μl

[0127] Oligo(dT) 18 1.0μl

[0128] 2×ES Reaction Mix 10.0μl

[0129] ES Reverse Transcriptase 1.0μl

[0130] RNase-free Water to 20.0μl

[0131] Gently suck and beat to mix, centrifuge slightly, keep warm at 42°C for 50min, and heat at 70°C for 15min. Then store at -20°C for later use.

[0132] 3. 5′ tailing reaction of cDNA

[0133] (a) Reaction system:

[0134] cDNA 20.0μl

[0135] 5×TdT Buffer 10.0μl

[0136] 0.1% BSA 5.0 μl

[0137] 100mM dCTP 1.0μl

[0138] TdT 1.0 μl

[0139] wxya 2 O Up to 50.0μl

[0140] (b) Incubate at 37°C for 30 minutes.

...

Embodiment approach ( 2

[0203] Embodiment (2): See SEQ.ID.NO.1 and SEQ.ID.NO.2 for the sequence of the novel cotton anti-fungal disease-related gene GhMPK7.

Embodiment approach ( 3

[0204] Embodiment (three): the construction of expression vector

[0205] (1) According to the nucleotide sequence of the isolated GhMPK7 gene, design primers:

[0206] Forward primer: 5′ TCTAGA ATGGCGACGCTTGTGGAGC-3′

[0207] The underlined part is the Xba I restriction site

[0208] Reverse primer: 5' GTC GAC CCTAAGCATTGGCAGATACAGCC-3′

[0209] The underlined part is the Sal I restriction site

[0210] The cDNA obtained by reverse transcription of the total RNA of young cotton leaves was used as a template for PCR reaction.

[0211] (2) Take 4 μl of the PCR product and connect it to the pMD18-T Simple vector, and the operation steps are carried out according to the instructions of the pMD18-T Simple Vector product of TaKaRa Company. Then the ligation product was transformed into Escherichia coli DH5α competent cells, and cultured overnight on LB solid medium containing ampicillin (100 mg / L). Pick the white colonies and culture them in LB liquid medium for 3 hours, a...

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Abstract

The invention relates to a novel cotton fungal disease-resistant gene GhMPK7 and application thereof, which belong to the field of molecular biology and biotechnology. In the invention, a gossypium hirsutum mitogen-activated protein kinases gene is obtained by adopting homology-based cloning and RACE technology. A cDNA sequence of the gene is positioned below a CaMV35S promoter to construct plant expression vectors and convert nicotiana benthamiana so that the gene is excessively expressed in tobacco. Experiments show that transgenic tobacco has high resistance to fungal diseases, in particular to colletotrichum nicotianae and alternaria alternate. When the gene is applied to crops such as wheat, corns and cottons, the fungal resistance can be improved and the yield and the quality can be improved. Thus, the gene has significant economical and social benefits.

Description

1. Technical field [0001] The invention relates to the cloning and recombination of a novel cotton anti-fungal disease-related gene GhMPK7 and the analysis and application of the anti-fungal disease ability of transgenic tobacco, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] Fungal diseases are the main diseases that limit the yield and quality of crops. They occur to varying degrees around the world every year, often leading to a decline in crop quality, a large reduction in production, or even a failure of harvest, which has brought significant economic losses to agricultural production. Fungal diseases generally have the characteristics of wide distribution, severe damage, wide host range, and multiple transmission routes, so it is relatively difficult to prevent and control them. At present, comprehensive management measures are mainly adopted, including breeding disease-resistant varieties and improving cultivation and...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/10C12N15/82A01H5/00
Inventor 郭兴启张良于菲菲石静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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