Screening method for using xylose isomerase genes for peanut genetic transformation

A xylose isomerase and genetic transformation technology, which is applied in the field of peanut molecular breeding, can solve problems such as inability to utilize xylose, achieve the effects of high transformation efficiency and avoid potential safety hazards

Inactive Publication Date: 2012-05-02
QINGDAO AGRI UNIV
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  • Abstract
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Problems solved by technology

Xylose is the main component of hemicellulose. There are some filamentous fungi, yeasts and bacteria that utilize xylose in nature, but ma

Method used

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  • Screening method for using xylose isomerase genes for peanut genetic transformation
  • Screening method for using xylose isomerase genes for peanut genetic transformation
  • Screening method for using xylose isomerase genes for peanut genetic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Establishment of a screening method using xylose isomerase gene for peanut genetic transformation

[0031] 1. Cloning of xylA gene

[0032] Design primers according to the xylA gene sequence in GenBank (Accession No. X04691), and add xho ⅠRestriction sites (italics):

[0033] The upstream primer is 5ˊ- CTCGAG ATGGAGTTCAATATGCAAGCCTA-3',

[0034] The downstream primer is 5ˊ- CTCGAG ATTATTTGTCGAACAGATAATGGTTT-3'.

[0035] The genomic DNA of Escherichia coli DH-5α strain was used as a template for PCR reaction to amplify the xylA gene and clone it into the pMD18-T vector (purchased from Treasure Biotech Dalian Engineering Co., Ltd.) to obtain the T-xylA recombinant plasmid.

[0036] 2. Construction of plant expression vector pCAMBIA1301-xylA

[0037] use xho Ⅰ Digest the pCAMBIA1301 plasmid (purchased from Shanghai Xipiji Biotechnology Co., Ltd.), remove the hygromycin-B-phosphotransferase gene (hygromycin-B-phosphotransferase, Hpt), and use xho Ⅰ D...

Embodiment 2

[0052] Embodiment 2, utilizing the xylose screening method to test the transformation of the glucanase gene Glu

[0053] The screening method using xylose isomerase gene of the present invention for peanut genetic transformation specifically comprises the following steps:

[0054] 1. Construction of a recombinant plasmid containing the target gene: Insert the target gene β-1,3-glucanase gene (Glu) (accession number M58462.1) into the plant expression vector pCAMBIA1301-xylA to obtain a recombinant plasmid containing the target gene Glu pCAMBIA1301-xylA-Glu.

[0055] 2. Transform the recombinant plasmid containing the target gene into peanut

[0056] The obtained recombinant plasmid pCAMBIA1301-xylA-Glu was transformed into peanut embryo leaflet explants by the Agrobacterium-mediated method (the transformation method was the same as in Example 1).

[0057] 3. Transfer the transformed embryo leaflet explants to somatic embryo induction medium for culture

[0058] The transfor...

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Abstract

The invention provides a screening method for using xylose isomerase genes for peanut genetic transformation. The screening method comprises the following steps of: constructing a recombinant vector with target genes; transforming the recombinant vector into peanut embryo lobule explants; transferring the transformed embryo lobule explants to an induction culture medium with sucrose and xylose for culturing until embryos are induced; transferring the embryos to a germination culture medium for culturing until seedlings are induced; performing PCR(polymerase chain reaction) detection on regrowth to obtain transgene positive plants; and transplanting the transgene positive plants to fields by a grafting method. According to the screening method, the culture medium with the 5 g/L of sucrose and the 10 g/L of xylose is used for culturing, so that the transgene plants can be screened, and hidden danger possibly caused by screening by using antibiotics is avoided, and the transformation efficiency is high, and the screening method is a safe and high-efficient screening method.

Description

technical field [0001] The invention relates to the field of peanut molecular breeding, in particular to a screening method for using xylose isomerase gene in peanut genetic transformation. Background technique [0002] Peanut is an important oil crop and economic crop in my country, and plays an important role in agriculture and even the entire national economy. However, due to the narrow genetic basis and poor stress resistance of peanut cultivars, they are particularly susceptible to various pests and diseases, which seriously affect their yield and quality. With the development of biotechnology, the use of transgenic technology to improve peanut cultivars has received extensive attention from researchers. The use of antibiotic genes and herbicide resistance genes for screening in the process of peanut genetic transformation may have adverse effects and damages on the environment and human health. Therefore, the use of uncontroversial biosafety marker genes to construct...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68A01H4/00A01H5/00
Inventor 乔利仙丁霄隋炯明王晶珊刘文平
Owner QINGDAO AGRI UNIV
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