Antioxidative protein from rubber tree as well as encoding gene and application of antioxidative protein

A coding and genetic technology, applied in the field of genetic engineering, can solve problems such as clogged milk ducts and rubber tree dead skin

Active Publication Date: 2014-04-23
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ROS can destroy the yellow body membrane in the latex of the rubber tree, causing the latex to solidify in situ, cau...

Method used

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  • Antioxidative protein from rubber tree as well as encoding gene and application of antioxidative protein
  • Antioxidative protein from rubber tree as well as encoding gene and application of antioxidative protein
  • Antioxidative protein from rubber tree as well as encoding gene and application of antioxidative protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, acquisition of rubber tree antioxidant protein HbAOP and its coding gene

[0062] According to the Hevea latex transcriptome deep sequencing database, the target fragment was selected, specific primers were designed, and then the cDNA sequence containing the complete reading frame was finally obtained by using the rapid amplification of cDNA ends (RACE).

[0063] The specific method is as follows:

[0064] (1) EST fragment cloning

[0065] Specific primers were designed as follows:

[0066] P1 (Forward): 5'-ATGGCTAATAAAAATGCTGA-3';

[0067] P2 (reverse): 5'-TCCATCAAAGCAACTGGAGT-3';

[0068] Using Heveabrasiliensis variety Reyan 7-33-97 (cultivated by the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences, and the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences has long-term seedlings for sale) cDNA obtained by reverse transcription of latex RNA random primers as a template, P1 and P2 ar...

Embodiment 2

[0086] Embodiment 2, research on HbAOP gene expression pattern in rubber tree

[0087] 1. Analysis of HbAOP tissue expression pattern

[0088] Using the cDNA randomly reverse-transcribed from the leaves, bark, latex, female flowers, and male flowers of Heveabrasiliensis variety Reyan 7-33-97 as a template, the HbAOP gene-specific primers HbAOP-QF and HbAOP-QR were used Bioengineering (Dalian) Co., Ltd. PremixExTaqII (TliRNaseHPlus) (catalog number RR820Q) for real-time fluorescent quantitative PCR. 18SrRNA was used as the internal reference, and the primers were 18SQF and 18SQR.

[0089] Primers for amplifying the HbAOP gene:

[0090] HbAOP-QF: 5'-GCTGAGTTGCAACGCCTTAC-3' (position 556-575 of SEQ ID NO: 2);

[0091] HbAOP-QR: 5'-ACAATCTTTTCCACGCCCCTC-3' (reverse complement of positions 622-641 of Sequence 2).

[0092] Primers for amplifying the internal reference 18SrRNA gene:

[0093] 18SQF: 5'-GCTCGAAGACGATCAGATACC-3';

[0094] 18SQR: 5'-TTCAGCCTTGCGACCATAC-3'.

[00...

Embodiment 3

[0106] Example 3, the acquisition of HbAOP transgenic strain and its antioxidant function verification

[0107] 1. Construction and identification of recombinant expression vector pEASY-E1-HbAOP

[0108] Use pEASY-E1 vector to construct the prokaryotic expression vector of HbAOP gene, the specific method is as follows:

[0109] The forward primer HbAOP-F and the reverse primer HbAOP-R were designed according to the HbAOP gene sequence (sequence 2), and the open reading frame of the HbAOP gene was amplified by PCR using the pMD18-HbAOP plasmid DNA obtained in Example 1 as a template.

[0110] HbAOP-F: 5'-ATGGCTGCAGCTACTTCTCT-3' (position 37-56 of Sequence 2);

[0111] HbAOP-R: 5'-CTAAACACCTGCTGTAGCTT-3' (reverse complement of positions 1691-1710 of SEQ ID NO: 2).

[0112] Reaction program: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 10 min.

[0113] After the reac...

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Abstract

The invention discloses an antioxidative protein from a rubber tree as well as an encoding gene and application of the antioxidative protein. The antioxidative protein provided by the invention is as shown in (a) or (b), wherein (a) is a protein which has an amino acid sequence as shown in a sequence 1 in a sequence table; (b) is a protein which is formed by substituting and/or deleting and/or adding one or more amino acid residues, has antioxidant activity and is derived from the sequence 1. Experiments prove that the encoding gene HbAOP of the antioxidative protein provided by the invention has the highest expression quantity in rubber tree leaf blades, and the expression of the gene is controlled by hydrogen peroxide, drought, low temperature and sodium chloride. The antioxidant activity of Escherichia coli can be improved through the prokaryotic expression of the gene HbAOP in the Escherichia coli, which proves that the gene HbAOP has the antioxidation. The gene HbAOP can be transferred into a plant or microorganism to improve the scavenging capacity to active oxygen, so that the oxidative stress resistance is improved.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a protein with antioxidant function derived from rubber trees, its coding gene and its application. Background technique [0002] Under normal physiological conditions, the production and removal of reactive oxygen species (Reactive Oxygen Species, ROS) in plants are in a state of dynamic balance. Stress conditions such as drought, low temperature, high temperature, salt stress, and heavy metals can disrupt the metabolic balance of ROS in plant cells, resulting in the accumulation of a large amount of ROS in the body. Excessive accumulation of ROS can not only directly attack the unsaturated fatty acids in the membrane system, lead to membrane lipid peroxidation and the production of malondialdehyde (MDA), which will destroy the membrane structure and reduce the fluidity of the membrane, and can also cause base mutations , DNA strand breaks and protein damage. Ultimately, it ca...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/21A01H5/00
CPCC07K14/415C12N15/8271C12N15/8273
Inventor 邓治聂智毅李德军
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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