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A Screening Method for Using Xylose Isomerase Gene in Peanut Genetic Transformation

The technology of a xylose isomerase and a screening method, which is applied in the field of peanut molecular breeding, can solve problems such as inability to utilize xylose, and achieve the effects of high transformation efficiency and avoiding potential safety hazards.

Inactive Publication Date: 2016-06-29
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 many plant cells cannot utilize xylose, so it is difficult to use safety marker genes such as xylose gene In plants to select transformants

Method used

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  • A Screening Method for Using Xylose Isomerase Gene in Peanut Genetic Transformation
  • A Screening Method for Using Xylose Isomerase Gene in Peanut Genetic Transformation
  • A Screening Method for Using Xylose Isomerase Gene in Peanut Genetic Transformation

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Effect test

Embodiment 1

[0028] Example 1. Establishment of a screening method using xylose isomerase gene for peanut genetic transformation

[0029] 1. Cloning of xylA gene

[0030] Design primers according to the xylA gene sequence in GenBank (Accession No. X04691), and add xho ⅠRestriction sites (italics):

[0031] The upstream primer is 5ˊ- CTCGAG ATGGAGTTCAATATGCAAGCCTA-3',

[0032] The downstream primer is 5ˊ- CTCGAG ATTATTTGTCGAACAGATAATGGTTT-3'.

[0033] 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.

[0034] 2. Construction of plant expression vector pCAMBIA1301-xylA

[0035] 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

[0050] Embodiment 2, utilizing the xylose screening method to test the transformation of the glucanase gene Glu

[0051] The screening method using xylose isomerase gene of the present invention for peanut genetic transformation specifically comprises the following steps:

[0052] 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.

[0053] 2. Transform the recombinant plasmid containing the target gene into peanut

[0054] 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).

[0055] 3. Transfer the transformed embryo leaflet explants to somatic embryo induction medium for culture

[0056] The transfor...

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Abstract

The invention provides a screening method for using xylose isomerase gene in peanut genetic transformation, which includes constructing a recombinant vector containing the target gene; transforming the recombinant vector into peanut embryo leaflet explant; explanting the transformed embryo leaflet The somatic embryos were transferred to the induction medium supplemented with sucrose and xylose for cultivation until the somatic embryos were induced; the somatic embryos were transferred to the germination medium for cultivation until the seedlings were induced; the regenerated seedlings were detected by PCR and positive for the transgene was obtained Plants; the transgenic positive plants were transplanted into the field by grafting. The present invention uses the substratum that has added the sucrose of 5g / L and the xylose of 10g / L to cultivate, and non-transformant can't become seedling because can not utilize xylose growth to be suppressed, and transformant can regenerate seedling because of being able to utilize xylose, Therefore, transgenic plants can be screened out, avoiding potential safety hazards that may be caused by using antibiotics for screening; the transformation efficiency is high, and it is a safe and 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
Inventor 乔利仙于明洋孙明明丁霄隋炯明刘文平陈立涛王晶珊
Owner QINGDAO AGRI UNIV
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