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Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants

A technology of peroxidase and ascorbic acid is applied in the field of genetic engineering to achieve the effect of improving stress resistance

Inactive Publication Date: 2011-08-24
AGRI BIOTECH RES CENT OF SHANXI PROVINCE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the APX gene research of the ancient and important economic tree species in China - jujube. So far, there has been no report of isolating the APX gene from jujube and applying it to improve plant stress resistance

Method used

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  • Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants
  • Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants
  • Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: jujube tree ascorbate peroxidase gene wxya the acquisition

[0028] In spring, fruiting branches (Ziziphus jujuba Mill. HUPING) with a length of about 2 mm (Ziziphus jujuba Mill. The mRNA was isolated and purified from the obtained total RNA, and a cDNA library was constructed using a cDNA library construction kit (purchased from Invitrogen, USA, catalog number: 18248-039). The library cDNA was ligated with the cloning vector pSPORT1, and the ligated product was transformed into E. coli DH5α competent cells, smeared on LB plates containing ampicillin resistance, cultured overnight at 37°C, screened recombinant plasmids with blue and white spots, randomly selected some positive plasmids, and entrusted Shanghai Sangong to the plasmids that proved to have inserted fragments after enzyme digestion sequencing.

[0029]The sequencing results were compared and analyzed using NCBI (www.ncbi.nlm.nih.gov / ) online analysis software, and the results showed that o...

Embodiment 2

[0032] Example 2: wxya Construction of Gene Plant Expression Vector

[0033] 1. Experimental materials

[0034] 1.1 Vectors and strains

[0035] Plant expression vector pEZR(K)-LNY, Escherichia coli ( E. coli )DH5α, carrying wxya The recombinant plasmid pSPORT1-ZjAPX of the gene was preserved by the Plant Cell and Embryology Laboratory of the Biotechnology Research Center of Shanxi Academy of Agricultural Sciences.

[0036] 1.2 Reagents and media

[0037] restriction endonuclease EcoR I. Sma I. Hind III. Bam H Ⅰ. DNA Ligation Kit, DL2000 DNA Marker, and Agarose Gel DNA Fragment Purification Kit were all purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[0038] Kanamycin (100mg / ml)

[0039] Phenol:chloroform:isoamyl alcohol (V:V:V=25:24:1)

[0040] Glucose solution (1M)

[0041] EDTA(0.5M, pH8.0)

[0042] 10%SDS(pH7.2)

[0043] NaOH(2M)

[0044] Tris-HCl (1M, pH8.0)

[0045] Sodium acetate (3M, pH5.2)

[0046] TE solution (10mM Tris-HCl, 1mM EDTA,...

Embodiment 3

[0109] Example 3: Agrobacterium-mediated wxya Gene Transformation of Arabidopsis

[0110] 1. Experimental materials

[0111] 1.1 Plant material

[0112] Wild-type Arabidopsis seeds were provided by the Plant Cell and Embryology Laboratory of the Biotechnology Research Center of Shanxi Academy of Agricultural Sciences, and the culture substrate was purchased from Horticultural Crops Research Institute of Shanxi Academy of Agricultural Sciences.

[0113] 1.2 Vectors and strains

[0114] The plant expression vector pEZR(K)-LNY-ZjAPX and Agrobacterium tumefaciens LBA4404 were preserved by the Plant Cell and Embryology Laboratory of the Biotechnology Research Center of Shanxi Academy of Agricultural Sciences.

[0115] 1.3 Reagents and Media

[0116] Kanamycin was purchased from Shanghai Sangong, and reagents used in YEB and 1 / 2MS medium were purchased from Tianjin Tianda Chemical Industry.

[0117] YEB medium:

[0118] Peptone 5g / L, beef extract 5g / L, yeast extract 1g / L, sucr...

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Abstract

The invention relates to the technical field of genetic engineering, in particular relating to a jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants. The nucleotide sequence of the jujube tree ascorbate peroxidase gene and the amino acid sequence of the coding protein of the jujube tree ascorbate peroxidase gene are shown in sequence tables of SEQ ID NO:1 and SEQ ID NO:2. In the invention, the jujube tree ascorbate peroxidase gene is obtained to construct a plant expression vector successfully transferred into an arabidopsis strain, and the salt tolerance of the jujube tree ascorbate peroxidase gene is evaluated, thereby proving that the jujube tree ascorbate peroxidase gene can be used for improving the salt tolerance of the plants. In addition, a prokaryotic expression vector constructed by the invention can be used for realizing induction and expression in escherichia coli. According to the invention, the first-time separation of the jujube tree ZjAPX (Ascorbate Peroxidase) gene is realized, and the gene can be used as a target gene transferred to the plants so as to improve the stress resistance, especially the salt tolerance of the plants, and has guiding significance in improving varieties of the plants, thereby providing test data and laying a theoretical basis for sufficiently utilizing the excellent-resistance gene ofjujube trees to improve the stress resistance of the plants.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a jujube ascorbate peroxidase gene and its application in improving plant stress resistance. Background technique [0002] Ascorbate peroxidase (APX), also known as vitamin C peroxidase, exists in higher plants, eukaryotic algae and some cyanobacteria, and APX activity is also detected in some insects. In higher plants, the ascorbic acid (AsA)-glutathione (GSH) cycle plays an extremely important role in scavenging reactive oxygen species. Among them, ascorbate peroxidase (APX) is the key enzyme in AsA-GSH cycle. The biggest feature of APX involved in active oxygen scavenging is that it has extremely high specificity for the substrate AsA, and can play a role at a lower level of AsA. The functional study of ascorbate peroxidase showed that APX played a significant role in improving the stress resistance of plants. Therefore, cloning the plant APX gene and enhancing ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/08A01H5/00
Inventor 孟玉平曹秋芬王振华郝子琪
Owner AGRI BIOTECH RES CENT OF SHANXI PROVINCE
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