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 inhibition, low production cost, growth and yield infection, etc.

Inactive Publication Date: 2012-07-25
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 (1): a kind of cloning method of cotton mitogen-activated protein kinase gene GhMKK5

[0129] 1. RNA extraction: use TRIZOL kit to extract total cotton RNA

[0130] 2. Synthesis of cDNA first-strand (using Transgen's 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 sucking and mixing, water bath at 42 °C for 30 min, 85 °C water bath for 10 min, cooled on ice for 5 min, lightly centrifuged, and stored 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] 1 μl of 100mM dCTP

[0144] TdT 1μl

[0145] ddH 2 O Up to 50μl

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

[0147] (c) Add 100 μl of absolute ethanol, and precipitate at -20°C...

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) Design primers according to the nucleotide sequence of the isolated cotton mitogen-activated protein kinase gene:

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

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

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

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

[0218] The PCR reaction was carried out using the above-mentioned forward and reverse primers using the full-length sequencing plasmid of the gene as a template.

[0219] (2) Take 4 μl of the PCR product and connect it to the pMD18-T Simple vector. The operation steps are carried out according to the instructions of the pMD18-T Simple Vector product of TaKaRa company. The ligation product was then transformed into E. coli DH5α competent cells and cultured on LB solid medium containing kanamycin (50 mg / L) overnight. Pick the white coloni...

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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 andquality 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 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 major bacterial crop diseases in my country, and the diseases are widely distributed and can infect a variety of plants. Infected plants often have a wide range of disease sites (roots, stems, and leaves can all be diseased), high mortality, and strong transmission, 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 has been paid more and more attention by people. The prevention and control measures of early bacterial diseases mainl...

Claims

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Application Information

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Patent Type & Authority Patents(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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