Fusion proteins, recombinant bacteria, and exosporium fragments for plant health

A fusion protein and protein fragment technology, applied in the field of fusion protein, can solve problems such as low survival rate and protein degradation

Pending Publication Date: 2022-01-04
BAYER CROPSCI LP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, previous attempts to introduce peptides, enzymes, and other proteins into soil to induce such beneficial effects on plants have been hampered by the low survival of enzymes, proteins, and peptides in soil
Furthermore, the ubiquity of naturally occurring proteases in soil can lead to the degradation of proteins in soil

Method used

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  • Fusion proteins, recombinant bacteria, and exosporium fragments for plant health
  • Fusion proteins, recombinant bacteria, and exosporium fragments for plant health
  • Fusion proteins, recombinant bacteria, and exosporium fragments for plant health

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0510]Example 1. Construction of Bacillus cereus family members displaying endopolygalacturonase

[0511] To construct a member of the Bacillus cereus family displaying the endopolygalacturonase of SEQ ID NO: 227, by combining the pUC57 plasmid (containing the ampicillin resistance cassette and the ColE1 origin of replication) with pBC16 from Bacillus cereus -1 plasmid (containing tetracycline resistance gene, repU replication gene and oriU origin of replication) was fused to generate pSUPER plasmid. Note that SEQ ID NO: 227 is identical to SEQ ID NO: 210 except that SEQ ID NO: 227 contains an extra cysteine ​​at its carboxy terminus. This 5.8 kb plasmid is replicable in both E. coli and Bacillus spp. and can be selected by conferring resistance to β-lactam antibiotics in E. coli and resistance to tetracycline in Bacillus spp. The base pSUPER plasmid was modified by inserting a PCR-generated fragment incorporating the BclA promoter (SEQ ID NO: 149), the start codon, amino aci...

Embodiment 2

[0514] Example 2. Canadian rape (canola) plant growth promotion research

[0515] Clear trays (5" x 5" x 2 1 / 2") were covered with a 5" x 5" VersaPak (Anchor Paper Company, St. Paul, MN) cushioning material. In each tray, place fifty canola seeds and treated with 50 mL of diluted whole broth so that each tray received at least 2 x 10 7 CFU of BT013A wild type strain or recombinant strain displaying endopolygalacturonase load ("EPG strain") as described in Example 1. Untreated control trays received 50 mL sterile water. One hundred seeds were tested for each strain, and untreated controls were placed in two trays. Place the trays in random order on the setting 450 μmol m -2 sec -1 Photosynthetic Photon Flux Density (PPFD), 14 hr light (28°C) / 10 hr dark (18°C) growth chamber rack for 21 days with daily rotation to minimize location effects. Watering was carried out daily from day 7 until the end of the experiment. No fertilizers or growth improvers are used.

[0516] Obse...

Embodiment 3

[0517] Example 3. Corn and soybean water stress research

[0518] Clear trays (8" x 8" x 3") were filled with sand to about 1 inch from the top and hydrated with 150 mL of water, with a fertilizer concentration of about 238 ppm N (measured as parts per million of nitrogen) Peters 20 - 20-20 all-purpose fertilizer (TheScotts Company, Marysville, OH) for improvement. Plant 16 corn and soybean seeds in four rows 1 inch deep, four seeds in each row. Subsequent saturate each with 50 mL of the above whole fermentation broth sample pallets such that each pallet accepts at least 2 x 10 7 CFU of whole broth cultures of BT013A wild-type strain or EPG strain. Untreated control trays received 50 mL sterile water. Place the trays in random order on the setting 450 μmol m -2 sec -1 PPFD (Photosynthetic Photon Flux Density), 14 hrs light (28°C) / 10 hrs dark (18°C) growth chamber rack for 14 days with daily rotation to minimize location effects. Watering was performed daily for eight da...

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Abstract

The present invention relates to a fusion protein having a targeting sequence, exosporium protein, or exosporium protein fragment that targets the fusion protein to the exosporium of a recombinant Bacillus cereus family member and a pectinase enzyme, wherein the pectinase is a pectate lyase from Bacillus spp. having any of SEQ ID NOs: 213-217 and 222-226 or a polygalaturonase from Aspergillus niger or certain Bacillus species that can have any of SEQ ID NOs: 210-212, 218-221, and 227. The present invention also provides a recombinant Bacillus cereus family member that expresses such fusion protein and exosporium fragments derived from such recombinant Bacillus cereus family member. Methods of using such recombinant Bacillus cereus family members or exosporium fragments derived therefrom for plant growth promotion are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 820,789, filed March 19, 2019, the entire contents of which are hereby incorporated by reference in their entirety. [0003] field of invention [0004] The present invention relates to a fusion protein comprising a targeting sequence, an exine protein or an exine protein fragment which targets the fusion protein to a recombinant Bacillus cereus ) family members of the spore wall (exosporium). The fusion protein also comprises a pectinase, including pectate lyase or polygalacturonase. The present invention also relates to a recombinant Bacillus cereus family member expressing the fusion protein, an exine fragment derived from the recombinant Bacillus cereus family member, and a preparation containing the recombinant Bacillus cereus family member or the exine wall fragment . Plant seeds treated with a recombinant Bacillus cereus family member...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C07K14/38A01N63/22A01N63/34
CPCC07K14/32A01N63/22A01N63/34A01N63/50C12N9/2402A01P21/00C07K2319/00C12N9/88C12Y302/01015C12Y402/02002
Inventor J·奥古斯汀D·柯蒂斯E·M·亨利S·K·霍顿A·拉马斯L·P·马纳瓦兰V·P·托马斯B·汤普森
Owner BAYER CROPSCI LP
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