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Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof

A technology of mannose phosphate and isomerase, which is applied in the field of biotechnology and plant genetic engineering, can solve problems such as safety concerns, insufficient attention to PMI safety, adverse effects of transformed receptor genomes, etc., to eliminate doubts, The effect of reducing potential security risks and addressing potential threats

Inactive Publication Date: 2015-07-08
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the widely used phosphomannose isomerase is isolated from prokaryotic Escherichia coli, because people have not realized that the phosphomannose isomerase isolated from prokaryotic E. Adverse effects on the receptor genome may also cause concerns about its safety, or people have not paid enough attention to the safety of this PMI isolated from prokaryotic E. coli
Therefore, there is an urgent need in this area for the higher plant source PMI screening marker gene with better safety, but there is no report of the PMI screening marker gene of higher plant source at present

Method used

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  • Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof
  • Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof
  • Phosphate mannose isomerase (PMI) gene OsPMI2 originating from oryza sativa and application thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1——OsPMI2 gene acquisition and cloning

[0044] The sequence is obtained in rice based on the PMI protein sequence of Escherichia coli. The present invention extracts the RNA corresponding to the sequence of the present invention in rice (Oryza sativa), and reverse transcribes it into cDNA; according to the CDS (coding sequence, coding sequence) sequence of OsPMI2, design gene-specific cloning primers, forward primer 5' -ATGGAGGACCCGCCGCCGCCGC-3', reverse primer 5'-TTAACTGAAGAATCTGCTGTTT-3'; then use cDNA as a template for PCR amplification.

[0045] Recover the target fragment amplified by PCR, the target fragment length is 1200bp, connect it to PGEM-T-Easy carrier (purchased from Promega Company, operate according to the instruction manual of the carrier), transform Escherichia coli XL-Blue competent cells according to the heat shock method; Then, positive clones were obtained by colony PCR screening. The identified positive clones were submitted to Invit...

Embodiment 2

[0047] Embodiment 2——The construction of the prokaryotic expression vector of OsPMI2 gene

[0048] By designing OsPMI2 prokaryotic expression primers, the forward primer 5'- CTCGAG ATGGAGGACCCGCCGCCGCCGC-3' (XhoI restriction site is underlined), reverse primer 5'- CTCGAG TTAACTGAAGAATCTGCTGTTT-3' (the underline is the XhoI restriction site), using the PGEM-T-OsPMI2 recombinant plasmid as a template, carry out PCR amplification, reclaim the target fragment of PCR amplification and the pGEX-6P-1 expression vector ( (purchased from GE Company) was connected to obtain the prokaryotic expression vector pGEX-OsPMI2 fused with the GST (glutathione-S-transferase) fragment, and transformed into Escherichia coli expression strain BL21.

Embodiment 3

[0049] Embodiment 3——OsPMI2 enzyme activity analysis

[0050] Line the BL21 strain containing the prokaryotic expression vector pGEX-OsPMI2, pick a single clone and inoculate it in 3 mL of ampicillin-containing LB liquid medium (see Table 1 for the Agrobacterium culture medium without agar), and culture overnight at 37°C with shaking ( 200r / min). The next day at room temperature, centrifuge at 5000r / min for 5min, discard the supernatant, resuspend the pellet with 3mL of fresh LB liquid medium, inoculate it in LB liquid medium containing ampicillin at a ratio of 1:100, culture with shaking at 37°C for 3h (200r / min), until OD600 (Optical density 600nm, 600nm absorbance value) reaches 0.6-1.0, add IPTG (isopropyl thiogalactoside, isopropyl thiogalactoside) to a final concentration of 1mM, and continue shaking culture at 37°C for 6h (200r / min).

[0051] 250mL culture with 80mL Bind / Wash Buffer (composition is 43mM Na 2 HPO 4 , 14.7mM KH 2 PO 4 , 1.37M NaCl, 27mM KCl, pH7.3...

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Abstract

The invention provides a phosphate mannose isomerase (PMI) gene OsPMI2 originating from a plant. The PMI gene OsPMI2 is separated and cloned from oryza sativa. A nucleotide sequence of the PMI gene OsPMI2 is shown in SEQ ID NO:1. The invention also provides a prokaryotic expression vector containing OsPMI2, an enzyme activity analysis method of PMI, an expression cassette containing OsPMI2, a plant expression vector and an application of the expression cassette and the expression vector to plant genetic transformation. Oryza sativa cell transformation is successfully achieved by utilizing the constructed plant expression vector of the gene OsPMI2 and taking mannose as a screening agent. The PMI gene originating from the plant is successfully separated and cloned. Originating from the plant (oryza sativa), the PMI gene does not pose a potential hazard to human. The PMI gene can be used to replace PMI from escherichia coli, thus reducing potential safety risks.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to the isolation, cloning and application of a phosphomannose isomerase gene OsPMI2 from rice (Oryza sativa). Background technique [0002] In transgenic technology, marker genes are often used to screen transformed cells or tissues. The potential safety risk of screening marker genes is an important aspect of public concern about transgenic technology. The existing transgenic technology mainly uses antibiotic resistance genes as selection markers. The antibiotic marker gene is transferred into the target crop together with the inserted target gene, which is used to help screen and identify transformed cells, tissues and regenerated plants in plant genetic transformation. The marker gene itself has no safety problems. However, this type of gene may enter the intestinal tract with food, and there is a potential ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12Q1/533C12N15/84C12N5/10
Inventor 李浩杨剑波魏鹏程李莉李娟杨亚春马卉秦瑞英
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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