Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Phosphomannose isomerase gene OsPMI1 originated 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 the problems of adverse effects on the transformed receptor genome, safety concerns, and insufficient attention to the safety of PMI, so as to solve potential threats , Eliminate doubts and reduce potential safety risks

Inactive Publication Date: 2015-07-29
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
View PDF1 Cites 4 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Phosphomannose isomerase gene OsPMI1 originated from oryza sativa and application thereof
  • Phosphomannose isomerase gene OsPMI1 originated from oryza sativa and application thereof
  • Phosphomannose isomerase gene OsPMI1 originated from oryza sativa and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1——OsPMI1 gene acquisition and cloning

[0038] The sequence is obtained from the rice genome based on the PMI protein sequence of Escherichia coli. The RNA of rice (Oryza sativa) was extracted and reverse-transcribed into cDNA; according to the CDS (coding sequence, coding sequence) sequence of OsPMI1, gene-specific cloning primers were designed, forward primer 5'-ATGGCCGGCCCTACTCCTCTCTC-3', reverse primer 5'-TTAATTGAAGAATCTGCTATTG-3'; then use cDNA as a template for PCR amplification. This primer has a better cloning effect on PMI.

[0039] Recover the target fragment amplified by PCR, the target fragment length is 1287bp, 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 Invi...

Embodiment 2

[0041] Embodiment 2——The construction of the prokaryotic expression vector of OsPMI1 gene

[0042] By designing OsPMI1 prokaryotic expression primers, the forward primer 5'- GGATCC ATGGCCGGCCCTACTCCTCTCTC-3' (the underline is the BamHI restriction site), reverse primer 5'- CTCGAG TTAATTGAAGAATCTGCTATTG-3' (the underline is the XhoI restriction site), using the PGEM-T-OsPMI1 recombinant plasmid as a template, carry out PCR amplification, recover the target fragment amplified by PCR and pGEX-6P-1 digested with BamHI and XhoI The expression vector (purchased from GE) was ligated to obtain the prokaryotic expression vector pGEX-OsPMI1 fused with the GST (glutathione-S-transferase) fragment, and transformed into Escherichia coli expression strain BL21.

Embodiment 3

[0043] Embodiment 3——OsPMI1 enzyme activity analysis

[0044] Line the BL21 strain containing the prokaryotic expression vector pGEX-OsPMI1, 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).

[0045] 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a phosphomannose isomerase gene OsPMI1 originated from oryza sativa. The nucleotide sequence of the phosphomannose isomerase gene OsPMI1 is as shown in SEQ ID NO:1. The invention further provides a prokaryotic expression vector containing the OsPMI1. The invention further provides an enzyme activity analysis method for the phosphomannose isomerase. Besides, the invention provides an expression box containing the OsPMI1 and a plant expression vector, as well as application of the expression box and the expression vector in the aspect of plant genetic transformation. According to the invention, the OsPMI1 gene is adopted to build the plant expression vector, mannose is used as a selective agent, and the transformation of oryza sativa cells is successfully realized; the phosphomannose isomerase gene originated from plant is successfully separated and cloned, no potential hazard is brought as the phosphomannose isomerase gene comes from plants (oryza sativa), and the phosphomannose isomerase gene originated from oryza sativa can be used for replacing the colibacillus-originated phosphomannose isomerase, so as to reduce the potential security risk.

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 OsPMI1 from rice (Oryza sativa). Background technique [0002] Transgenic technology is an effective method for directional improvement of plants developed in the early 1980s. This technology mainly introduces the target gene into the receptor through methods such as Agrobacterium-mediated and gene gun transformation, and obtains stably expressed transformants after marker screening and molecular detection, and realizes rapid and directional improvement of target traits. It has a wide range of available gene resources, improved High efficiency and other advantages. Transgenic technology has provided a new path and opened up new space for crop yield improvement, quality improvement and resistance enhancement. The development and applicatio...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/61C12N9/90C12N15/82C12N5/10A01H5/00
Inventor 魏鹏程杨剑波李莉杨亚春李娟胡磊马卉秦瑞英许蓉芳李浩
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com