Clone of cotton mitogen activated protein kinase gene GhMAPK16 and application thereof

A protein kinase gene, mitogen activation technology, applied in the field of molecular biology and biology, can solve the problems of long cycle, heavy workload, long breeding cycle and so on

Inactive Publication Date: 2009-12-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Measures to enhance crop fitness by improving soil structure are long-term and costly
Measures to improve the salt tolerance of plants by improving cultivation and management techniques have had little effect
However, due to the long breeding cycle,

Method used

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  • Clone of cotton mitogen activated protein kinase gene GhMAPK16 and application thereof
  • Clone of cotton mitogen activated protein kinase gene GhMAPK16 and application thereof
  • Clone of cotton mitogen activated protein kinase gene GhMAPK16 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment approach ( 1

[0142] Embodiment (1): A method for cloning cotton mitogen-activated protein kinase gene GhMAPK16

[0143] 1. Extraction of RNA: Extraction of cotton total RNA using TRIZOL kit

[0144] 2. Synthesis of the first strand of cDNA

[0145] Add the following reagents to a 0.25ml centrifuge tube:

[0146] mRNA 4μl

[0147] oligo d(T) primer 2μl

[0148] DEPC-H 2 O 6μl

[0149] After mixing by suction and mixing, put in a water bath at 65°C for 5 minutes, cool on ice for 5 minutes, centrifuge gently, and add the following reagents:

[0150] 5×First-Strand Buffer 4μl

[0151]Rnasin Ribonuclease Inhibitor 1μl

[0152] 10mM dNTP 1μl

[0153] EasyScript Reverse Transcriptase 1μl

[0154] 0.1M DTT 2μl

[0155] Gently tap to mix well, centrifuge slightly, incubate at 42°C for 1 hour, then store at -20°C for later use.

[0156] 3. 5′ tailing reaction of cDNA

[0157] (a) Reaction system:

[0158] cDNA 20μl

[0159] 5×TdT Buffer 10μl

[0160] 0.1% BSA 5 μl

[0161] 100mM dCTP...

Embodiment approach ( 2

[0227] Embodiment (2): See SEQ.ID.NO.1 and SEQ.ID.NO.2 for the sequence of cotton mitogen-activated protein kinase gene GhMAPK16.

Embodiment approach ( 3

[0228] Embodiment (three): the construction of expression vector

[0229] (1) according to the nucleotide sequence of the isolated cotton mitogen-activated protein kinase gene, design primers:

[0230] Forward primer: 5′- TCTAGA GGGGCTTCCTGTTTGATGCC-3′

[0231] The underlined part is the Xba I restriction site

[0232] Reverse primer: 5′- GTC GAC GGGGCTTCCTGTTTGATGCC-3′

[0233] The underlined part is the Sal I restriction site

[0234] The cDNA obtained by reverse transcription of the total RNA of young cotton leaves was used as a template for PCR reaction.

[0235] (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 product pMD 18-T Simple Vector produced by 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 colonies and culture t...

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Abstract

The invention relates to clone and recombination of a cotton mitogen activated protein kinase gene as well as salt resistance function analysis and application thereof, belonging to the fields of molecular biology and biotechnology. In the invention, total RNA is extracted from primary leaves of the cotton, and reverse transcription is carried out to obtain obtains cDNA. A degenerate primer is designed according to a conservative amino acid sequence of the mitogen activated protein kinase gene in the other plants to carry out a conventional polymerase chain reaction, a PCR product is connected with a pMD18-T carrier to transform a colibacillus DH5 alpha competent cell, a recombinant is selected to carry out sequence analysis, and the full-length cDNA is obtained through a rapid amplification technology of 3'end and 5' end. A plant positive expression vector is further constructed to transform the tobacco cultivated variety NC89, the transgenic tobacco has the obvious salt resistance capacity so as to transform the gene into crops such as cottons, wheat, corns, and the like, thereby improving the salt resistance capacity of the crops and increasing the yield and the quality of the crops, and having important economy value and social value.

Description

1. Technical field [0001] The invention relates to the cloning and recombination of a cotton mitogen-activated protein kinase gene GhMAPK16 and the analysis and application of the salt-resistant ability of transgenic tobacco, belonging to the fields of molecular biology and biotechnology. 2. Background technology [0002] The severe shortage of fresh water, land and other resources in my country's crop production has seriously restricted the sustainable development of my country's agriculture. Nearly one-third of the arable land is affected by different degrees of salinization, and the area of ​​salinized arable land Still increasing year by year. In addition, there are more than 1.5 billion mu of wasteland and tidal flats in our country that are saline-alkali land. According to conservative estimates, environmental stresses such as salinity, low temperature and other environmental stresses reduce the output of major crops in my country by more than 15% of the total output e...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12P19/34C12N15/82
Inventor 郭兴启张良石静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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