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Phosphomannose isomerase gene with plant origin and application of phosphomannose isomerase gene

A mannose phosphate, plant-derived technology, applied in the isolation, cloning and application of mannose phosphate isomerase gene ChlaPMI, can solve safety concerns, the safety of PMI has not attracted enough attention, and the adverse effects of transforming the recipient genome And other issues

Inactive Publication Date: 2015-04-22
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • Phosphomannose isomerase gene with plant origin and application of phosphomannose isomerase gene
  • Phosphomannose isomerase gene with plant origin and application of phosphomannose isomerase gene
  • Phosphomannose isomerase gene with plant origin and application of phosphomannose isomerase gene

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Embodiment 1

[0037] Embodiment 1 - the acquisition and cloning of ChlaPMI gene

[0038]The sequence is based on the bacterial PMI protein sequence, compared with the homologous sequence in the genome sequence of Chlamydomonas that can utilize mannose (www.ncbi.nlm.nih.gov), and the one with the highest homology is ChlaPMI protein sequence. The RNA of Chlamydomonas reinhardtii was extracted and reverse-transcribed into cDNA; according to the CDS (coding sequence) sequence of ChlaPMI, gene-specific cloning primers were designed, forward primer 5'-ATGCTGCAGCTTCGCTGTGCTG-3', reverse primer 5'-CACCTCCGCCTCTCCAGCCGTC-3'; PCR amplification was performed using cDNA as a template.

[0039] Recover the target fragment amplified by PCR, the target fragment length is 1095bp, 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,...

Embodiment 2

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

[0042] By designing ChlaPMI prokaryotic expression primers, the forward primer 5'- GGATCC ATGCTGCAGCTTCGCTGTGCTG-3' (the underline is the BamHI restriction site), reverse primer 5'- CTCGAG CTCCGCCTCTCCAGCCGTC-3' (the underline is the XhoI restriction site), using the PGEM-T-ChlaPMI 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 the prokaryotic expression vector pGEX-ChlaPMI fused with the GST (glutathione-S-transferase) fragment, and transformed into Escherichia coli expression strain BL21. At the same time, the pGEX-6P-1 empty vector and the pGEX-PMI containing the Escherichia coli PMI expression vector were also transformed into the Escherichia coli expression strain BL21.

Embodiment 3

[0043] Embodiment 3——ChlaPMI activity analysis

[0044] The BL21 bacterial strain containing the prokaryotic expression vectors pGEX-ChlaPMI, pGEX-PMI and pGEX-6P-1 empty vector was drawn, and a single clone was picked to 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...

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Abstract

The invention provides a phosphomannose isomerase gene ChlaPMI with the plant origin. The nucleotide sequence of the phosphomannose isomerase gene ChlaPMI is shown as SEQ ID NO: 1. The invention further provides a prokaryotic expression vector with ChlaPMI, and the prokaryotic expression vector can be used for identifying the ChlaPMI proteometabolism mannose activity. Based on the prokaryotic expression vector, the ChlaPMI proteometabolism mannose activity is identified so that it can be identified that the prokaryotic expression vector can carry out metabolism on the mannose. The invention provides an expression box with the ChlaPMI, a plant expression vector and application of expression box and expression vector in the plant genetic transformation application. The plant expression vector established through the ChlaPMI gene is utilized, the mannose serves as the screening reagent, and rice cells are successfully converted. The phosphomannose isomerase gene with the plant origin is successfully separated and cloned, and due to the fact that the phosphomannose isomerase gene comes from the plant (Chlamydomonas reinhardtii), the phosphomannose isomerase gene is an environmentally-friendly natural substance, and potential danger cannot be caused to the human beings. The phosphomannose isomerase gene can be used for replacing phosphomannose isomerase from the Escherichia coli.

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 ChlaPMI from Chlamydomonas reinhardtii. 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. [0003] However, the e...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/63C12N15/61C12N15/82C12N5/10A01H5/00A01H5/02A01H5/06A01H5/10
CPCC12N9/90C12N15/8205C12N15/8245C12Y503/01007
Inventor 李浩杨剑波魏鹏程李莉李娟杨亚春
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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