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Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast

A technology of mannose isomerase and isomerase, applied in the fields of botanical equipment and methods, horticultural methods, genetic engineering, etc.

Inactive Publication Date: 2012-11-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, the phosphomannose isomerase genes used in the above studies are all from Escherichia coli, and there is no report on using the phosphomannose isomerase (PMI) gene of Saccharomyces cerevisiae as a screening marker for genetic transformation of plants such as rice

Method used

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  • Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast
  • Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast
  • Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast

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Embodiment 1

[0063] Example 1 Cloning of Saccharomyces cerevisiae phosphomannose isomerase PMI gene

[0064] From Saccharomyces cerevisiae (Saccharomyces cerevisiae) HB52 (gifted by Professor Hao Bo, School of Life Science and Technology, Huazhong Agricultural University, see: Kong Lin, Hao Bo, Yu Ziniu, Zinc-rich yeast selection and cultivation technology, Food and Biotechnology Journal , 2006, Volume 25, Issue 6) extracted genomic DNA and cloned the phosphomannose isomerase PMI gene. The specific steps are as follows: Inoculate a ring of fresh Saccharomyces cerevisiae into 20mL YPD liquid medium, YPD medium contains: 2% tryptone, 1% yeast extract, 2% glucose, 30°C, 180r / min shaking culture for 18~ 22h. The cells were collected by centrifugation, resuspended with normal saline, and the supernatant was discarded by centrifugation. Put the collected bacteria into a 5mL centrifuge tube, add 1mL of lysis buffer (50mmol / L Tris-HCl pH 8.0, 180mmol / L EDTA pH 8.0, 1% SDS, prepared now) for ever...

Embodiment 2

[0070] The construction of the pPMI carrier of embodiment 2 PMI gene

[0071] The transformation vector containing the PMI gene constructed by the applicant, which is named pPMI, is transformed from the plasmid pCAMBIA1303 (see the construction flow chart figure 2 ). pCAMBIA1303 contains a kanamycin resistance gene, a hygromycin resistance gene linked to a 35S promoter and a CaMV terminator, and a GUS-GFP fusion protein linked to a 35S promoter and a NOS terminator between the left and right T-DNA borders Gene. Escherichia coli containing the pCAMBIA1303 plasmid was inoculated in LB medium (10% tryptone, 5% yeast extract, 10% NaCl), cultivated overnight at 150 rpm at 37° C., extracted the plasmid, and used chloroform: isoamyl alcohol (volume ratio of 24 : 1) Purify the plasmid. Digest the purified plasmid overnight at 37°C to excise the hygromycin resistance gene. The PCR product was also digested with Xho I overnight after purification. According to pCAMBIA1303 plasmid ...

Embodiment 3

[0072] Example 3 Rice Genetic Transformation and Screening

[0073] The recipient plant of the present invention is rice (Oryza Sativa L) variety Mudanjiang No. 8, and the seeds of this variety are provided by Professor Lin Yongjun of the Rice Group of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University. The method of genetic transformation of the present embodiment is: first remove the chaff, soak with 70% ethanol for 1 min, then sterilize with 0.15% mercuric acid for 20 min, then wash with sterile water for 3-4 times and inoculate to the rice callus induction medium , 26 ℃ dark culture to induce callus. After 35-40 days, take the vigorous, light yellow granular callus and transfer it to the subculture medium, and subculture in the dark at 26°C. The embryogenic callus that has been subcultured for 1-2 times can be Agrobacterium-mediated genetic transformation experiments. The pale yellow callus granules with strong viability and dense text...

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Abstract

The invention belongs to the technical field of plant genetic engineering and relates to a method for screening a transgenic paddy plant by a phosphomannose-isomerase (PMI) gene of yeast. The method comprises the following steps of primer designing, PCR amplification, carrier construction, genetic transformation, screening and identification. A PMI gene obtained by cloning of brewer's eucaryon yeast is used as a selection marker. Through an agrobacterium-mediated transformation method, the PMI gene is introduced into a paddy acceptor. Transformed paddy callus is screened in mannose mediums having different concentration gradients so that a transgenic paddy plant is obtained. The method has the advantages that in plant transformation and screening, antibiotics and herbicides are not used; and the transgenic paddy plant screened by the method does not contain an antibiotic gene or a herbicide gene and is safe for the environment and a human body.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and in particular relates to a method for screening rice transgenic plants by using yeast mannose isomerase gene. Background technique [0002] Plant genetic engineering began in the 1970s. In 1983, tobacco was successfully introduced as the first transgenic plant. At present, at least 35 families and more than 200 kinds of plants have been successfully transgenic. Transgenic research provides breeders with a new way to improve crops. It can transfer beneficial genes from plants, animals or microorganisms into plants to improve the yield, quality or stress resistance of the original varieties. [0003] The screening system for plant transgenes often uses antibiotic genes or herbicide-resistant genes as selection markers, and their expression products can relieve the toxicity of antibiotics and herbicide-resistance to cells. For example, hpt gene as a selection marker can eliminate the t...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/82C12N1/21C12Q1/68A01H4/00A01H5/00
Inventor 张方东唐永严刘良玉王涛张美冬郑用琏
Owner HUAZHONG AGRI UNIV
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