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The invention discloses cotton polyamine oxidase GhPAO2 gene and application thereof

A kind of polyamine oxidase, cotton technology, applied in the field of genetic engineering

Active Publication Date: 2013-12-04
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current research on plant polyamine oxidase is mainly concentrated on model plants such as Arabidopsis thaliana, rice, tobacco, and a few crops such as corn, barley, etc. No relevant reports have been found on cotton

Method used

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  • The invention discloses cotton polyamine oxidase GhPAO2 gene and application thereof
  • The invention discloses cotton polyamine oxidase GhPAO2 gene and application thereof
  • The invention discloses cotton polyamine oxidase GhPAO2 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Cloning and Sequence Analysis of Cotton Polyamine Oxidase GhPAO2 Coding cDNA

[0025] 1.1 Cloning of cotton polyamine oxidase GhPAO2 cDNA

[0026] Using the cDNA that was not inoculated with Verticillium dahliae as the driver, and the induced 'Jimian 20' cDNA as the tester, follow the subtraction kit BD PCR Select from Clontech Company TM The instructions for the cDNA Subtraction Kit perform suppression subtraction hybridization. The cDNA obtained by SSH was connected with pGEM-T Easy Vector (Beijing Tianwei Times Technology Co., Ltd.), and then transferred into E. coli competent cells Top10 by heat shock method. The positive clones were selected and sequenced to construct upland cotton varieties resistant to Verticillium wilt SSH library. Based on an EST sequence in the library, a cDNA sequence was obtained through homologous comparison and bioinformatics analysis of the EST database on the NCBI website, which was presumed to be the cDNA sequence encoding ...

Embodiment 2

[0048] Example 2 Expression Analysis of Cotton Polyamine Oxidase GhPAO2 in Escherichia coli

[0049] 1.1 Construction of prokaryotic expression recombinant vector

[0050] The pCold TF plasmid and the pMD18-T plasmid (pMD18-GhPAO2) containing the mORF of the GhPAO2 gene were subjected to a double enzyme digestion reaction with KpnI and XbaI respectively. The enzyme digestion reaction system is as follows:

[0051]

[0052] Carry out agarose gel electrophoresis on the digested products, and recover the corresponding digested fragments respectively according to the instructions of the EZ Spin Column DNA Gel Extraction Kit UN1Q-10 column DNA gel recovery kit from Shanghai Bioengineering Co., Ltd., and then incubate at 16°C After ligation by T4 DNA ligase for 12 hours, 10 μL ligation reaction system is as follows:

[0053]

[0054] The ligation product was transformed into competent cells DH5α. After heat shock transformation, the positive clones were screened and sent to S...

Embodiment 3

[0062] Example 3 Analysis of Cotton Polyamine Oxidase GhPAO2 Catalytic Properties in Vitro

[0063] 1.1GhPAO2 is a flavoprotein with FAD as prosthetic group

[0064] Existing studies have proved that PAO is a type of flavoprotein with FAD as a prosthetic group. Through non-covalent bonding, FAD acts as an electron donor to oxidize oxygen molecules in polyamine oxidase and stimulate the oxidation activity of PAO. Sequence comparison analysis shows that the N-terminus of the GhPAO2 protein has a FAD binding fingerprint pattern. In order to verify whether the purified protein uses FAD as a prosthetic group, the present invention takes the purified protein solution, adds 5% chloroform and shakes vigorously, and then centrifuges at 12,000 rpm for 10 minutes at 4°C. , take the supernatant and continuously measure the absorption value at 300-540nm on the Shimadzu UV-1750 UV-Vis spectrophotometer, which shows that two typical FAD protein absorption peaks appear at 380 and 460nm ( Im...

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Abstract

The invention discloses a cotton polyamine oxidase GhPAO2 gene and application thereof. The sequence of the cotton polyamine oxidase GhPAO2 gene is shown in SEQ ID No. 3. According to the invention, the root tissue of Jimian 20 is taken as raw material; the whole-length cDAN sequence of the cotton polyamine oxidase GhPAO2 gene is cloned through bioinformatics and the RT-PCR technology; the cotton polyamine oxidase GhPAO2 gene is constructed to a prokaryotic expression vector and expresses a heterologous protein in an escherichia coli Rosetta (DE3) successfully. An in-vitro experiment proves that purified cotton polyamine oxidase GhPAO2 GhPAO2 is a flavoprotein taking FAD as a prothetic group and has the activity of a spermidine oxidase and a spermine oxidase. A bacterial strain capable of expressing the cotton polyamine oxidase GhPAO2 and a contrast bacterial strain containing an empty carrier are treated with sodium chloride solution with different concentrations; with the same treatment, the quantity of cells of the transgenic bacterial strain is remarkably increased when compared with that of the cells of the contrast bacterial strain, which shows the expression of the cotton polyamine oxidase GhPAO2 in the escherichia coli improves the salt tolerance of the host bacteria.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the cotton polyamine oxidase GhPAO2 gene and its application. Background technique [0002] Cotton is a "pioneer plant" in the development and utilization of saline wasteland, and the development and utilization of saline wasteland for cotton planting is an important way to alleviate the conflict between grain and cotton caused by insufficient cultivated land and to achieve sustainable development of cotton production. In my country, the coastal saline-alkali land alone can expand the cotton planting area by more than 1.3 million hm 2 . However, the salt-tolerant ability of cotton is limited, especially in recent years, the degree of soil salinization has been increasing, resulting in the decline of insect-resistant cotton, yield and quality, which has seriously hindered the development of the cotton industry. Therefore, new salt-tolerant varieties are cultivated. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/63C12N1/21C12N15/82A01H5/00C12R1/19
Inventor 马峙英李亚栋孙艳香王省芬张艳
Owner HEBEI AGRICULTURAL UNIV.
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