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Hevea brasiliensis polyphenol oxidase, its coding gene and application

A technology of polyphenol oxidase and coding gene, which is applied in the direction of oxidoreductase, application, genetic engineering, etc., and can solve problems such as the protein sequence of the enzyme coding gene that has not been seen

Inactive Publication Date: 2012-10-24
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Even so, there is no report about the enzyme coding gene and its protein sequence so far

Method used

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  • Hevea brasiliensis polyphenol oxidase, its coding gene and application
  • Hevea brasiliensis polyphenol oxidase, its coding gene and application
  • Hevea brasiliensis polyphenol oxidase, its coding gene and application

Examples

Experimental program
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Embodiment 1

[0024] Example 1 Separation of polyphenol oxidase HbPPO2

[0025] HbPPO2 can be isolated from cells and tissues such as buds, leaves, seeds, callus and suspension cells. The present invention preferably recommends the use of suspension cells for two reasons: 1). Suspension cells can be obtained in large quantities by culturing; 2). In suspension cells Only HbPPO2 and another polyphenol oxidase, which was also expressed in milk duct cells, were present, and HbPPO2 was significantly induced after the suspension cells were treated with suction, while the other polyphenol oxidase was almost disappeared. Suspension cells can be derived from different tissues of rubber trees. The inner integument and immature anthers of seeds are preferred. Taking the inner integument as an example, the process is as follows:

[0026] Callus induction: add 3% sucrose, 1.0 mg / L 2, 4-D (2, 4-dichlorophnoxyacetic acid, 2, 4-dichlorophenoxyacetic acid), 1.0 mg / L BAP (6-benzylaminopurine, 6-benzyladenin...

Embodiment 2

[0030] Example 2 Cloning of polyphenol oxidase HbPPO2 coding gene

[0031] Since there are only two kinds of polyphenol oxidase in the suspension cells of the rubber tree, we used them as materials for RNA extraction and PCR template preparation; since there has been no report about the polyphenol oxidase encoding gene in the rubber tree, we based on other species The sequences of the conserved Cu-binding region in PPO were designed to incorporate primers, and two differential gene fragments were successfully amplified from cDNA templates derived from suspension cells by PCR technology; on this basis, we used the rapid amplification technology RACE of cDNA ends Successfully obtained the 3' and 5' sequences of HbPPO2; by designing primers at the 3' and 5' ends of HbPPO2, using genomic DNA from leaves and cDNA from suspension cells as templates, we successfully amplified the genome of this gene for the first time and cDNA sequences. The specific process is as follows:

[0032]...

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Abstract

The invention discloses a hevea brasiliensis polyphenol oxidase. The gene is intronless, has a cDNA sequence with a length of about 1869 bp, a sequence of SEQ ID NO: 1 shown in a sequence table, and is named HbPPO2. The HbPPO2 codes 592 amino acids, has a sequence of SEQ ID NO: 2 shown in the sequence table, and can be acidic protein a molecular weight of 67 kDa, an isoelectric point of 6.66, and an electrical charge of -0.4. The enzyme at least exists in buds, leaves, seeds, calluses and suspension cells, and the protein level is induced at least by pathogen infection and mechanical damage. The enzyme or its derivative protein at least can be used in food and industrial fields to bleach or oxidize colored substances. Utilizing the antisense expression of part or all of the sequences of the gene can at least inhibit the browning phenomenon in hevea brasiliensis tissue culture. The functional derivative gene over-expression obtained through the gene at least can improve the disease and pest resistance of hevea brasiliensis.

Description

technical field [0001] The invention relates to a rubber tree polyphenol oxidase, its encoding gene and application. Background technique [0002] Polyphenol Oxidase (PPO) is a class of copper-containing oxidoreductase widely distributed in animals, plants, fungi and bacteria. at O 2 In the presence of PPO, PPO can catalyze the hydroxylation of monophenols to o-diphenols, and the oxidation of dihydric phenols to o-quinones. The generalized PPO includes laccase (laccase, EC.1.10.3.1), tyrosinase (tyrosinase, or monophenol oxidase, monophenolase, EC.1.14.18.1) and catechol oxidase (catechol oxidse, or bisphenol Oxidase, o-diphenoloxidase, EC.1.10.3.2). Generally speaking, PPO refers to the general designation of catechol oxidase and laccase. [0003] Catechol oxidase is the main PPO in plants, which is encoded by nuclear genes and exists in multiple gene families in most plants. PPO usually exists in the form of an inactive precursor, which is generally composed of an N-t...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N5/10A01H5/00
Inventor 邹智杨礼富王真辉袁坤
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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