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Gene GhMKK5 capable of improving bacterial resistance of transgenic crops and application thereof

A gene, protein kinase gene technology, applied in the field of molecular biology and biology, can solve the problems of low production cost, growth and yield infection, inhibition and so on

Inactive Publication Date: 2011-08-17
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the application of cotton MAPKK to improve the agronomic traits of crops at home and abroad.
[0004] As one of the most important economic crops in the world, cotton has low production cost and high application value, but its growth and yield are severely inhibited by pathogen infection

Method used

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  • Gene GhMKK5 capable of improving bacterial resistance of transgenic crops and application thereof
  • Gene GhMKK5 capable of improving bacterial resistance of transgenic crops and application thereof
  • Gene GhMKK5 capable of improving bacterial resistance of transgenic crops and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment ( 1

[0128] Embodiment (one): A kind of cloning method of cotton mitogen-activated protein kinase gene GhMKK5

[0129] 1. Extraction of RNA: Extraction of cotton total RNA using TRIZOL kit

[0130] 2. Synthesis of the first strand of cDNA (transgen company cDNA first strand synthesis kit method)

[0131] Add the following reagents to a 0.25ml centrifuge tube:

[0132] mRNA 5μl

[0133] Oligo d(T) 1μl

[0134] Buffer 10μl

[0135] EasyScript Reverse Transcriptase 1μl

[0136] RNAase free water 3μl

[0137] After mixing by pipetting, take a water bath at 42°C for 30 minutes, then a water bath at 85°C for 10 minutes, cool on ice for 5 minutes, centrifuge lightly, and store for later use.

[0138] 3. 5′ tailing reaction of cDNA

[0139] (a) Reaction system:

[0140] cDNA 20μl

[0141] 5×TdT Buffer 10μl

[0142] 0.1% BSA 5 μl

[0143] 100mM dCTP 1μl

[0144] TdT 1μl

[0145] wxya 2 O Up to 50μl

[0146] (b) Incubate at 37°C for 30 minutes.

[0147] (c) Add 100 μl of abso...

Embodiment ( 2

[0211] Example (2): See SEQ.ID.NO.1 and SEQ.ID.NO.2 for the sequence of cotton mitogen-activated protein kinase gene GhMKK5.

Embodiment ( 3

[0212] Embodiment (three): the construction of expression vector

[0213] (1) according to the nucleotide sequence of the isolated cotton mitogen-activated protein kinase gene, design primers:

[0214] Forward primer: 5′- TCTAGA CATTCATTTCCTTTTAGCTTCCTC-3′

[0215] The part underlined is the Xba Ⅰ restriction site

[0216] Reverse primer: 5′- GTC GAC CAGAAGATTTTTTCCCTAATCC-3′

[0217] The part underlined is the Sal Ⅰ restriction site

[0218] Using the full-length sequencing plasmid of the gene as a template, the above-mentioned forward and reverse primers were used for PCR reaction.

[0219] (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 kanamycin (50mg / L). Pick the white colo...

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Abstract

The invention relates to a gene GhMKK5 capable of improving bacterial resistance of transgenic crops and an application thereof, belonging to the fields of molecular biology and biotechnology. According to the invention, a cotton mitogen-activated protein kinase gene GhMKK5 is obtained by the homologous cloning and RACE technology; the cDNA (complementary deoxyribonucleic acid) of the gene is placed under a CaMV 35S starter to construct a plant expression vector; and nicotiana benthamiana is transformed so that the gene is over-expressed in tobacco. Experiments demonstrate that: the transgenic tobacco has obvious capability in resisting bacteriosis. By applying the gene to the crops such as transformed wheat, cotton and the like, the antibacterial capability can be enhanced, the yield and quality can be improved, and tremendous economic and social values can be realized.

Description

1. Technical field [0001] The invention relates to the cloning and recombination of a cotton mitogen-activated protein kinase kinase gene GhMKK5 and its application in improving the resistance of transgenic tobacco bacteria, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] Bacterial diseases are diseases caused by bacterial infection, and it is difficult to control them. There are 60-70 kinds of major bacterial crop diseases in my country, and the diseases are widely distributed and can infect many plants. Susceptible plants often have a wide range of diseased sites (roots, stems, and leaves can all be affected), high mortality, and strong dissemination, resulting in a large reduction in crop production or even extinction. Therefore, the prevention and control of bacterial diseases has become a worldwide problem and more and more people pay attention to it. The prevention and control measures of early bacterial diseases main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/82
Inventor 郭兴启孟飞张良李玉真吴长艾
Owner SHANDONG AGRICULTURAL UNIVERSITY
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