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Bird rape xanthomonas essential gene and coded protein and application thereof

A technology of Xanthomonas and essential genes is applied in the field of essential genes of Xanthomonas campestris and the proteins encoded by them, which can solve the problems that the biological function and application of genes are not proved by experiments.

Inactive Publication Date: 2012-04-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The nucleic acid sequence of Xanthomonas campestris pv. campestris, the pathogen of black rot in cruciferous plants (such as cabbage, cabbage, mustard, radish, Arabidopsis, etc.), has been reported by the genome sequencing project (Qian W, Jia YT, He YQ et al. 2005. Comparative and functional genomic analyzes of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Research, 15: 757-767.), although the project on genes in Xanthomonas campestris Analysis was performed, but the biological functions and applications of specific genes were not experimentally demonstrated

Method used

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  • Bird rape xanthomonas essential gene and coded protein and application thereof
  • Bird rape xanthomonas essential gene and coded protein and application thereof
  • Bird rape xanthomonas essential gene and coded protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, phoP is bacterial essential gene

[0040] 1. Construction of phoP stringent expression recombinant vector

[0041] Since the mutation of the essential gene will lead to bacterial cell death, the genetic method based on homologous recombination cannot be used to directly mutate and knock out the phoP gene. To mutate it, an additional copy of the phoP gene must be provided in the bacterial cell, and the transcription of the copy can be controlled by a stringently regulated promoter. On this basis, the phoP copy on the chromosome can be genetically manipulated.

[0042] For this purpose, primer 1 (5'-GTCGACATGCGTATCCTTTTGGTCGA-3') and primer 2 (5'-GGTACCTCAGCCTTCCGTACGCGGGA-3'), namely SEQ ID No.3 and SEQ ID No.4 in the sequence listing, were amplified by PCR method The full-length sequence (684bp) of the phoP gene of Xanthomonas campestris strain 8004 (hereinafter referred to as Xcc 8004). The amplification conditions were 94°C for 2min; 94°C for 30sec, 60...

Embodiment 2

[0047] (2) Example 2, PhoP is a response regulator protein

[0048] 1. PhoP-His 6 Expression and purification of recombinant proteins

[0049] According to bioinformatics prediction, PhoP contains Receiver domain and transcription factor domain, and is a possible bacterial response regulator protein. However, there is no experimental evidence for this speculation. Since the response regulator protein will be phosphorylated by chemical small molecules such as acetate phosphate, and can be phosphorylated by the corresponding histidine kinase. Therefore, the present invention expresses and purifies PhoP protein and analyzes its phosphorylation characteristics. Utilize primer 7 (5'-CATATGCGTATCCTTTTGGTCGA-3') and primer 8 (5'-CTCGAGGCCTTCCGTACGCGGGA-3'), that is, SEQ ID No. in the sequence listing, 9 and SEQ ID No.10, amplify the whole phoP by PCR method long clips. The amplification conditions were annealing at 94°C for 2min; 28 cycles of 94°C for 30sec, 60°C for 60sec, and ...

Embodiment 3

[0054] (3) Example 3, downregulation of phoP gene transcription in the absence of arabinose culture can significantly inhibit bacterial growth

[0055] The successful acquisition of the essential gene phoP knockout mutant makes it possible to artificially manipulate the expression of this gene and observe its effect on bacterial growth. Because in this mutant, phoP is placed under the control of the stringent arabinose promoter (pBAD-araC). If a certain amount of arabinose is added to the medium, the promoter can be induced to drive the transcription of phoP. If the culture medium does not contain arabinose, since the copy of phoP located on the recombinant vector pHM2-pBAD-phoP stops transcribing, and the copy of phoP on the chromosome has been deleted in-frame, it cannot regulate the normal growth of bacteria, which will lead to growth arrest or even death. like Figure 5 As shown, when Xanthomonas strains were grown in NYG rich nutrient medium (containing arabinose) to O...

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Abstract

The invention discloses a bird rape xanthomonas essential gene and coded protein and application thereof. The essential gene has one of the following nucleotide sequences: (1) a DNA (Deoxyribonucleic Acid) sequence of SEQ ID No.1 in a sequence table, and (2) polynucleotide for encoding the SEQ ID No.2 of an amino acid sequence in the sequence table. The protein coded by the essential gene has an amino acid sequence of SEQ ID No.2 in the sequence table. The essential gene or the coded gene thereof can be taken as a target for screening substance resisting bird rape xanthomonas. The essential gene and the coded protein thereof are essential for the growth of bird rape xanthomonas, so that the essential gene and the coded protein thereof can be taken as ideal and specific pesticide acting targets of bird rape xanthomonas. The crucifer black rot caused by bird rape xanthomonas is a severe hazard to the production of a large quantity of vegetables in the world, so that the research, the development and the application of the protein and the gene play a unique role in controlling the block rot.

Description

technical field [0001] The invention relates to an essential gene of Xanthomonas campestris and its encoded protein and application. Background technique [0002] Bacterial essential genes refer to the genes necessary to maintain the normal growth and reproduction of bacterial cells. Essential genes generally encode key molecules in cell metabolic activities and biochemical processes, such as key proteins in nucleic acid, amino acid, fat and cell wall synthesis, cell division, material transport, and gene expression regulation. Its mutation or inactivation will directly lead to growth arrest or death of bacterial cells. Therefore, essential genes are also ideal drug targets for the development of new antibiotics or antibacterial compounds, and the biological activity or stability of their encoded products can be used to screen possible antibacterial compounds. The two-component signal transduction system of bacteria is composed of histidine kinase and response regulator, w...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12Q1/68C07K14/21G01N33/68C12N15/70C12R1/64C12R1/19
Inventor 钱韦彭宝玉
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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