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Phosphomannose isomerase from chlorella variabilis 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 and adverse effects of transformed receptor genomes, and achieve the effect of eliminating safety concerns and solving potential threats

Active Publication Date: 2015-04-22
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 the prokaryotic Escherichia coli, which may have adverse effects on the genome of the transformed recipient and may also raise concerns about its safety

Method used

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  • Phosphomannose isomerase from chlorella variabilis and application thereof
  • Phosphomannose isomerase from chlorella variabilis and application thereof
  • Phosphomannose isomerase from chlorella variabilis and application thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1 - the acquisition and cloning of ChloPMI gene

[0039] The sequence is based on the bacterial phosphomannose isomerase protein sequence, compared with the homologous sequence in the mannose-utilizing Chlorella genome sequence (genome.jgi-psf.org), and the highest homology is obtained. is the ChloPMI protein sequence. The bacterial phosphomannose isomerase protein sequence is as follows:

[0040] MQKLINSVQNYAWGSKTALTELYGMENPSSQPMAELWMGAHPKSSSEVQNAAGDIVSLRDVIESDKSTLLGEAVAKRFGELPFLFKVLCAAQPLSIQVHPNKHNSEIGFAKENAAGIPMDAAERNYKDPNHKPELVFALTPFLAMNAFREFSEIVSLLQPVAGAHPAIAHFLQQPDAERLSELFASLLNMQGEEKSRALAILKSALDSQQGEPWQTIRLISEFYPEDSGLFSPLLLNVVKLNPGEAMFLFAETPHAYLQGVALEVMANSDNVLRAGLTPKYIDIPELVANVKFEAKPANQLLTQPVKQGAELDFPIPVDDFAFSLHDLSDKETTISQQSAAILFCVEGDATLWKGSQQLQLKPGESAFIAANESPVTVKGHGRLARVYNKL

[0041] Then, the RNA of Chlorella variabilis was extracted and reverse-transcribed into cDNA; according to the CDS (coding sequence) sequence of ChloPMI, gene-specific cloning p...

Embodiment 2

[0043] Embodiment 2——The construction of the prokaryotic expression vector of ChloPMI gene

[0044] By designing ChloPMI prokaryotic expression primers, the forward primer 5'- GGATCC ATGGCTGGAACGGCGACAGAGA-3' (the underline is the BamHI restriction site), reverse primer 5'- CTCGAG CTCAAAGGCCATTCCGTTG-3' (the underline is the XhoI restriction site), using the PGEM-T-ChloPMI recombinant plasmid as a template, carry out PCR amplification, recover the target fragment amplified by PCR and digest pGEX-6P-1 with BamHI and XhoI for expression The vector (purchased from GE) was ligated to obtain a prokaryotic expression vector pGEX-ChloPMI fused with a GST (glutathione-S-transferase) fragment, and transformed into Escherichia coli expression strain BL21. At the same time, pGEX-6P-1 empty vector and pGEX-PMI containing Escherichia coli phosphomannose isomerase expression vector were also transferred into Escherichia coli expression strain BL21.

Embodiment 3

[0045] Embodiment 3——ChloPMI activity analysis

[0046] Line the BL21 bacterial strain containing the prokaryotic expression vectors pGEX-ChloPMI, pGEX-PMI and pGEX-6P-1 empty vector, pick a single clone and inoculate the LB liquid medium (see Table 1 for the Agrobacterium culture medium without adding agar), Shake culture at 37°C overnight (200r / min). The next day, centrifuge at 6000r / min for 1min at room temperature, discard the supernatant, and resuspend the pellet with a small amount of sterile water. Take the resuspension and inoculate it into sterile phenol red chromogenic medium (1% peptone, 0.5% sodium chloride, 50mg / L phenol red, 30% mannose, pH 7.4) at a ratio of 1:50, and culture with shaking at 37°C ( 200r / min), observe the color change of the medium after 48h. If the strain has the ability to metabolize mannose, the medium will be acidified, the pH value will drop, and the color of the medium will gradually change from red at pH 7.4 to yellow. For the results o...

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Abstract

The invention provides phosphomannose isomerase from chlorella variabilis. The phosphomannose isomerase from the chlorella variabilis is named as ChloPMI. The invention further provides a prokaryotic expression vector containing the ChloPMI, and the prokaryotic expression vector can be used for identifying the proteometabolism mannose activity of the ChloPMI. The invention further provides an expression cassette containing the ChloPMI, a plant expression vector, application of the expression cassette and the application of the plant expression vector on the aspect of plant genetic transformation. The plant expression vector constructed by utilizing the ChloPMI uses mannose as a screening reagent and successfully achieves conversion of rice cells. The phosphomannose isomerase is successfully separated and cloned from the chlorella variabilis, due to the facts that the chlorella variabilis derives from the chlorella variabilis, is environmentally friendly and has no potential risks to the human body, application and popularization of transgenic products are facilitated, and doubts about transgenosis are cleared.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to the separation and cloning of a phosphomannose isomerase gene ChloPMI from chlorella (Chlorella variabilis); the construction of a prokaryotic expression vector containing ChloPMI and its identification of ChloPMI protein metabolism mannose activity The construction of the plant expression vector containing ChloPMI, and the application of ChloPMI in plant genetic transformation with ChloPMI as a screening marker. 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 direct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/63C12N15/61C12N15/82C12N5/10A01H5/00A01H5/02A01H5/06A01H5/10
CPCC12N9/90C12N15/8241C12Y503/01008C12N15/82C12N15/821C12N9/00C12N5/14C12N15/70C12N15/74
Inventor 李莉杨剑波魏鹏程李浩杨亚春李娟
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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