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Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof

A protein kinase gene, mitogen activation technology, applied in the field of molecular biology and biology

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

[0007] After searching, there is no report about the cotton yield-related gene GhMAPK6 in domestic and foreign literatures and patents

Method used

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  • Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof
  • Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof
  • Gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment approach ( 1

[0137] Embodiment (1): A method for cloning cotton mitogen-activated protein kinase gene GhMAPK6

[0138] 1. Extraction of RNA: Extraction of cotton total RNA using TRIZOL kit

[0139] 2. Synthesis of the first strand of cDNA (transgen company cDNA first strand synthesis kit method)

[0140] Add the following reagents to a 0.25ml centrifuge tube:

[0141] mRNA 5μl

[0142] Oligo d(T) 1μl

[0143] 2×Buffer 10μl

[0144] EasyScript Reverse Transcriptase 1μl

[0145] RNAase free water 3μl

[0146] After mixing by pipetting, put in a water bath at 42°C for 30 minutes, then in a water bath at 85°C for 5 minutes, cool on ice for 5 minutes, centrifuge lightly, and store for later use.

[0147] 3. 5′ tailing reaction of cDNA

[0148] (a) Reaction system:

[0149] cDNA 20μl

[0150] 5×TdT Buffer 10μl

[0151] 0.1% BSA 5 μl

[0152] 100mM dCTP 1μl

[0153] TdT 1μl

[0154] wxya 2 O Up to 50μl

[0155] (b) Incubate at 37°C for 30 minutes.

[0156] (c) Add 100 μl of absolute...

Embodiment approach ( 2

[0233] Embodiment (2): See SEQ.ID.NO.1 and SEQ.ID.NO.2 for the sequence of cotton mitogen-activated protein kinase gene GhMAPK6.

Embodiment approach ( 3

[0234] Embodiment (three): the construction of expression vector

[0235] (1) according to the nucleotide sequence of the isolated cotton mitogen-activated protein kinase gene, design primers:

[0236] Forward primer: 5′- ggatc cgtaagaatggaaggcggag-3′

[0237] The part underlined is the restriction site of BamH Ⅰ

[0238] Reverse primer: 5' gagctc tggactgatcactgctgca-3′

[0239] The part underlined is the Sac Ⅰ restriction site

[0240] Using the full-length sequencing plasmid of the gene as a template, the above-mentioned forward and reverse primers were used for PCR reaction.

[0241] (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 colony Pick ...

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Abstract

The invention relates to a gossypium hirsutum mitogen-activated protein kinas 6 (GhMAPK6) gene and application thereof, belonging to the fields of molecular biology and biotechnology. The GhMAPK6 gene is obtained by adopting homologous cloning and RACE [Rapid-Amplification of cDNA (complementary Deoxyribonucleic Acid) Ends] technology. The cDNA sequence of the gene is put under a CaMV 35S promoter to construct a plant expression vector, and nicotiana benthamiana is transformed to excessively express the gene in tobacco. An experiment proves that a transgenic tobacco plant has remarkably improved yield than a non-transgenic tobacco plant in the aspects of increase in the leaf area, increase in the tillering number, increase in the infruit number and increase in the ripening rate. Therefore, the gene has important realistic significance in improving the crop yield trait and increasing the crop yield by a gene engineering way according to the yield increasing property of the gene.

Description

(1) Technical field [0001] The invention relates to the cloning and recombination of a cotton mitogen-activated protein kinase gene GhMAPK6 and its application in improving the yield of transgenic tobacco, belonging to the fields of molecular biology and biotechnology. (2) Background technology [0002] As the world population continues to surge, people's demand for food is also increasing dramatically. At the same time, the wanton development of resources and environment by human beings has gradually destroyed the ecological balance and greatly affected the food production. In our country, with the continuous improvement of economic level and the continuous acceleration of urbanization and industrialization, the proportion of agricultural population and usable arable land is decreasing year by year. Therefore, how to increase food production to meet people's growing basic living material needs is the current Problems to be solved. [0003] The traditional methods to incre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/82
Inventor 郭兴启李玉真张良张伟吴长艾
Owner SHANDONG AGRICULTURAL UNIVERSITY
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