Construction and application of trans-Sm1-chit 42 trichoderma engineering bacteria

A technology of sm1-chit42, pcambia1300th-sm1-linker-chit42, applied in the application, fungicide, fungi and other directions, can solve the problem of no report, etc., achieve the effect of cucumber seed germination and plant growth promotion, and control of cucumber powdery mildew

Inactive Publication Date: 2019-03-01
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It has been reported to construct a single chitinase genetically engineered bacterium that transforms Trichoderma by transgenic methods, but the construction of a bivalent engineered bacterium with a small hydrophobic protein Sm1 derived from Trichoderma and a chit42 gene derived from Metarhizium anisopliae is difficult both at home and abroad. no report

Method used

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  • Construction and application of trans-Sm1-chit 42 trichoderma engineering bacteria
  • Construction and application of trans-Sm1-chit 42 trichoderma engineering bacteria
  • Construction and application of trans-Sm1-chit 42 trichoderma engineering bacteria

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Experimental program
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Effect test

Embodiment 1

[0050] Cloning of embodiment 1, Sm1 and Chit42 genes

[0051] Trichoderma T.harzianum T30 and Metarhizium anisopliae cultured on PDA for 7 days were used as materials for total RNA extraction. The total RNA of Trichoderma was extracted and reverse-transcribed by using Tiangen RNA Extraction Kit and Reverse Transcription Kit. Chit42 and Sm1 genes were based on the homologous target gene sequences JF345997 and XM_014104331.1 in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ), PCR primers were designed using Primer Premier 5.0 software, and Chit42 and Sm1 were cloned. Sm1 gene, primers were synthesized by Sangon (Shanghai) Bioengineering Co., Ltd.

[0052] SEQ ID No: 4 Chit42-F: 5'-ATGCCGTCGTTATTTGCTCAGT-3'

[0053] SEQ ID No: 5 Chit42-R: 5'-CTAAGCCATCTGCTTCCTCATAT-3'

[0054] SEQ ID No: 7 Sm1-F: 5'-CGCGGATCCATGCAACTGTCCAACA-3'

[0055] SEQ ID No: 8 Sm1-R: 5'-CCGCTCGAGGAGACCGCAGTTCT-3'

[0056] Total PCR reaction system 25 μL, template 1 μL, high-fidelity enzyme Premix St...

Embodiment 2

[0059] Embodiment 2, the construction of Sm1-Chit42 overexpression Trichoderma engineering bacteria

[0060] The fusion gene overexpression frame is composed of five parts, the promoter and terminator are trpC promoter and terminator, the primers are designed, and the templates are mixed successively. The two fusion gene expression frames are divided into fragment 1 and fragment 2, using SEQ ID No: 12 and SEQ ID No: 17 as primers, using SEQ ID No: 2 and SEQ ID No: 11 as templates to amplify fusion fragment 1; SEQ ID No: 18, SEQ ID No: 16 are primers, and fragment 2 is amplified with SEQ ID No: 2 and SEQ ID No: 6; using SEQ ID No: 12, SEQ ID No: 16 as primers to obtain the fragment 1. Fragment 2 is used as a template, and the overexpression frame ORF (that is, the Sm1-linker-chit42 fusion overexpression frame) is obtained by fusion and amplification. The overexpression cassette was constructed on the pCAMBIA1300th vector by restriction enzyme ligation.

[0061] SEQ ID No: 1...

Embodiment 3

[0072] Embodiment 3, fusion protein purification

[0073] The engineered bacterial strains cultured in liquid were taken, filtered by suction to remove free hyphae, and filtered again with a 0.22 μm bacterial filter to obtain a clear liquid containing extracellular eggs. Use an ultrafiltration centrifuge tube to retain substances with a molecular weight above 3KD, centrifuge at 4000g for 10min, collect the liquid in the filter tube, and absorb PBS buffer solution to wash the filter membrane four times to obtain the remaining protein on the filter membrane. The extract solution increases the protein concentration. The engineering strain protein has a His tag, and the Superdex20010 / 300GL high-performance liquid column and AKTA primeplus chromatographic system of GE Company were used for FPLC (fast protein liquid chromatography), with 0.4mL / min 1MTris-HCl (pH6.8) as the mobile phase, Analyze the collected proteins. Figure 6 The result shows that the present invention success...

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Abstract

The invention discloses a construction and an application of trans-Sm1-chit 42 trichoderma engineering bacteria. According to the invention, a Trichoderma harzianum (T.harzianum T30) bacterial strainis taken as a carrier; chitinase Chit42 gene and hydrophobin Sm1 gene with the function of activating disease resistance of plants are selected from the carrier; oriented molecular design is performedon overexpression of fusion gene according to the structural biologic and bioinformatic principle, so as to construct the Sm1-chit 42 trichoderma engineering bacteria capable of expressing fusion gene; the fermentation liquor is prepared into raw powder of engineered protein crude extract. The raw powder of engineered protein crude extract disclosed by the invention is capable of effectively preventing and controlling powdery mildew of cucumber and has the function of promoting growth of cucumber plant. Preventing effect can be promoted by spraying the engineered protein in a seedling stage.

Description

technical field [0001] The invention relates to the construction of a transgenic Sm1-chit42 Trichoderma engineering bacterium, the preparation method of a crude extract and its application in the prevention and treatment of cucumber powdery mildew and the like. Background technique [0002] Trichoderma is a beneficial fungus commonly found in soil and rhizosphere ecosystems. After Trichoderma colonizes the rhizosphere soil and roots of plants, it can induce the resistance of crops to infectious diseases and abiotic stress, and improve crops' absorption and utilization of soil nutrients. ability. On the other hand, after Trichoderma colonizes plant leaves, a series of cell wall degrading enzymes (such as chitinase Chit42, Chit33 and β-1,3-glucanase) can directly degrade the cell wall of the pathogenic bacteria it contacts to complete reparasitization. At the same time, Trichoderma can also secrete elicitor proteins, such as chitinase, hydrophobin and other elicitor proteins ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/15A01N63/04A01P3/00C12R1/885
CPCA01N63/30C07K14/37C07K2319/00C12N9/2402C12N15/80C12Y302/01014
Inventor 刘宏毅夏海陈捷刘健邵家华
Owner SHANGHAI JIAO TONG UNIV
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