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Phosphate mannose isomerase (PMI) gene OsPMI3 originating from oryza sativa and application thereof

A technology of mannose phosphate and isomerase, which is applied in the fields of biotechnology and plant genetic engineering, can solve the problem that there is no PMI screening marker gene report of higher plant origin, the safety of PMI has not been paid enough attention, and the adverse effects of transformation receptor genome To solve potential threats, eliminate doubts about the safety of genetically modified foods, and reduce potential safety risks

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

[0006] 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 OsPMI3 originating from oryza sativa and application thereof
  • Phosphate mannose isomerase (PMI) gene OsPMI3 originating from oryza sativa and application thereof
  • Phosphate mannose isomerase (PMI) gene OsPMI3 originating from oryza sativa and application thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 - the acquisition and cloning of OsPMI3 gene

[0046] The sequence is extracted from rice, and the invention extracts a predetermined segment from rice. The RNA of rice (Oryza sativa) was extracted and reverse-transcribed into cDNA; according to the CDS (coding sequence, coding sequence) sequence of OsPMI3, gene-specific cloning primers were designed, forward primer 5'-ATGACAGCAAGCAAAGAAACAG-3', reverse Primer 5'-TCAGCTGAAGAATCTGCTGTTC-3'; then use cDNA as a template for PCR amplification.

[0047] Recover the target fragment amplified by PCR, the target fragment length is 660bp, connect it to the PGEM-T-Easy carrier (purchased from Promega Company, operate according to the instruction manual of the carrier), and 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 Invitrogen for sequencing. The verified correct ...

Embodiment 2

[0049] Embodiment 2——The construction of the prokaryotic expression vector of OsPMI3 gene

[0050] By designing OsPMI3 prokaryotic expression primers, the forward primer 5'- GAATTC ATGACAGCAAGCAAAGAAACAG-3' (the underline is the EcoRI restriction site), reverse primer 5'- CTCGAG TCAGCTGAAGAATCTGCTGTTC-3' (the underline is the XhoI restriction site), using the PGEM-T-OsPMI3 recombinant plasmid as a template, carry out PCR amplification, recover the target fragment amplified by PCR and digest pGEX-6P-1 with EcoRI and XhoI to express The vector (purchased from GE) was ligated to obtain the prokaryotic expression vector pGEX-OsPMI3 fused with the GST (glutathione-S-transferase) fragment, and transformed into Escherichia coli expression strain BL21.

Embodiment 3

[0051] Embodiment 3——OsPMI3 enzyme activity analysis

[0052] Line the BL21 strain containing the prokaryotic expression vector pGEX-OsPMI3, pick a single clone and inoculate it in 3 mL of LB liquid medium containing ampicillin (see Table 1 for the Agrobacterium culture medium without adding 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).

[0053] 250mL culture with 80mL Bind / Wash Buffer (composition is 43mM Na 2 HPO 4 , 14.7mM KH 2 PO 4 , 1.37M NaCl, 27mM KC...

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Abstract

The invention provides a phosphate mannose isomerase (PMI) gene OsPMI3 originating from a plant. A nucleotide sequence of the PMI gene OsPMI3 is obtained after carrying out addition, deletion, replacement or insertion of one or a plurality of nucleotides on a nucleotide sequence shown in SEQ ID NO:1. The invention also provides a prokaryotic expression vector containing OsPMI3 and an enzyme activity analysis method of PMI. Oryza sativa cell transformation is successfully achieved by utilizing the constructed plant expression vector of the gene OsPMI3 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 OsPMI3 from rice (Oryza sativa). Background technique [0002] Transgenic technology has brought great convenience to the study of plant functions and molecular breeding. Currently, there are dozens of marker genes used in the study of transgenic plants, among which screening marker genes are often used to identify plant transformants, such as herbicide resistance genes and hygromycin resistance gene, etc. [0003] In plant genetic transformation, marker genes are often used to screen transformants containing target genes, and make them regenerate completely, and obtain transformed plants with improved genetic traits after differentiation. [0004] The potential safety risk of screening marker genes is an important aspect of public concern...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/70C12N15/84A01H5/00
Inventor 李浩杨剑波魏鹏程李娟李莉杨亚春许蓉芳秦瑞英胡磊马卉
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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