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Pseudomonas pseudoalcaligenes disinsection protein gene

A technology of insecticidal protein and insecticidal protein, applied in genetic engineering, plant gene improvement, peptide preparation method, etc., can solve the problem of low molecular weight of protein, and achieve the effect of improving expression

Inactive Publication Date: 2007-11-28
SICHUAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] In the research on the insecticidal protein of Pseudomonas alcaligenes, it has been confirmed that the molecular weight of the protein is low, and it is a kind of exotoxin protein (Zhang Zhang, Yang Zhirong, Zhu Wen, etc. Separation, purification and identification [J]. Acta Microbiology, 1998, 38: 57-62), but there is no report on the cloning of the Pseudomonas pseudoalcaligenes insecticidal protein gene and transgenic plants at home and abroad

Method used

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Experimental program
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Effect test

Embodiment 1

[0028] Isolation and Purification of Insecticidal Protein from Pseudomonas-like Pseudomonas

[0029] 1.1 Culture medium preparation

[0030] Inoculate Pseudomonas pseudoalcaligenes in broth medium (Zhou Deqing. Microbiology Experiment Manual [M], Shanghai Science and Technology Press, 1985), medium components (g / L): beef extract 5g, egg white 10g , NaCl 5g, pH 7.2. After culturing at 35°C for 12h, transfer to the same medium, 35°C, 120r / min, shaking culture for 36h for later use.

[0031] 1.2 Detection of Insecticidal Activity of Pseudomonas Alcaligenes Culture

[0032] The culture of Pseudomonas pseudoalcaligenes was centrifuged at 6000 g for 10 min at 4°C to obtain the bacterial culture supernatant and bacterial precipitate. The bacterial pellet was washed three times with deionized water and then homogenized. After the obtained homogenate was centrifuged at 10,000 g, the homogenate supernatant was collected, and its protein concentration was determined to be 43 mg / ml. Af...

Embodiment 2

[0049] Cloning and prokaryotic expression of insecticidal protein gene of Pseudomonas pseudoalcaligenes and insecticidal experiment.

[0050] 2.1 Construction of Pseudomonas alcaligenes genome library

[0051]Genomic DNA of Pseudomonas pseudoalcaligenes was extracted by CTAB method, and then a small amount of genomic DNA was taken for Sau3AI enzyme digestion pre-experiment to confirm that the generated random DNA fragments were concentrated in 2-10kb. Sau3AI is partially digested, and the digested products are separated by 0.5% agarose gel electrophoresis, and 2-10kb fragments are recovered from the agarose gel. The pUC19 plasmid (Norrander, J. et al., Gene (1983) 26: 101-106) was used as the cloning vector, and the BamHI site of the plasmid vector was used as the cloning site. When pUC19 was completely digested by BamHI and transformed into a linear molecule, the 5' phosphate groups at both ends of the linear molecule were removed with calf intestinal alkaline phosphatase to...

Embodiment 3

[0066] Insecticidal protein gene for alfalfa transformation and breeding of insect-resistant alfalfa

[0067] 3.1 Vector construction:

[0068] 3.1.1 Construction of vector pCAMBIA2301G

[0069] The construction process of the vector pCAMBIA2301G is shown in FIG. 2 . Firstly, the plant expression vectors pBI121 and pCAMBIA2301 were digested with EcoRI and HindIII. Then, recover pBI121 about 3Kb fragment, pCAMBIA2301 about 11Kb fragment, and use T 4 DNA ligase ligated to obtain the vector pCAMBIA2301G.

[0070] 3.1.2 Construction of vector pCAMBIA2301G-IP

[0071] The vector pSC1-1 was used as a template to PCR amplify the Pseudomonas pseudoalcaligenes insecticidal protein gene fragment, which was digested with XbaI and SacI to recover a 1.4kb fragment. At the same time, the vector pCAMBIA2301G was also digested with XbaI and SacI to recover a fragment of about 12Kb, and then ligated with the digested Pseudomonas-like insecticidal protein gene fragment to obtain the expres...

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Abstract

The present invention provides a kind of new pesticidal protein gene and the gene coded protein. The pesticidal protein gene is originated from Pseudomonas pseudoalcalligenes, and its coded protein has wide and powerful intoxicating effect on grasshopper and Orthoptera insects, so that the pesticidal protein gene may be used in preventing and controlling pest of crop. The present invention also provides the Pseudomonas pseudoalcalligenes pesticidal protein separating and purifying method, and one method of producing pesticidal transgenic plant.

Description

technical field [0001] The present invention relates to an insecticidal protein gene of Pseudomonas pseudoalcaligenes, which can be used for expression in host cells. Background technique [0002] In the past 30 years, many microbial insecticides with entomopathogenic microorganisms as the main components have been published one after another. Due to the different pathogenic microorganisms used by these microbial pesticides, there are great differences in the insecticidal mechanism. It has been proven that the insecticidal mechanism of bacterial insecticides is usually closely related to bacterial toxin proteins (Yu Ziniu, Production and Application of Bacillus thuringiensis [M]. Beijing: Agricultural Press 1993). Since the discovery of the first toxin protein, diphtheria toxin, in 1888, hundreds of bacterial toxins have been found in various bacteria. Among them, many bacterial toxin proteins have been studied in detail. [0003] So far...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/21C07K1/36C12N15/31A01H1/00
Inventor 杨志荣张杰赵建岳碧松
Owner SICHUAN UNIV
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