Tobacco S-phase kinase-associated protein 1 gene sequence and its coding protein sequence and application

A gene sequence and kinase protein technology, applied in the field of S-phase kinase-related protein 1 gene, can solve problems such as unclear specific pathways, and achieve obvious guiding effects

Inactive Publication Date: 2008-07-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, studies have found that the ubiquitin system mediated by SCF plays a role in biological reactions such as the jasmonic acid signaling

Method used

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  • Tobacco S-phase kinase-associated protein 1 gene sequence and its coding protein sequence and application
  • Tobacco S-phase kinase-associated protein 1 gene sequence and its coding protein sequence and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] 1. Cloning and sequence of the cDNA of the S-phase kinase-related protein 1 gene of tobacco (Sansheng tobacco)

[0014] 1) Spray tobacco leaves with 50 mg / L chitosan oligosaccharide, spray water as a control, take samples and extract total RNA for DDRT-PCR at 8h and 168h respectively, recover and subclone the differential fragments, and sequence to obtain the 3' end sequence. The homology of the fragment with the SKP1 (S-PHASEKINASE-ASSOCIATED PROTEIN1) gene of Nicotiana benthamiana is 82%.

[0015] 2) Obtain the 5' end of the SKP1 gene and the full length of the cDNA of Nicotiana solanifolia using a kit from CLONTECH, and then deduce the amino acid sequence.

[0016] 3) Nicotiana solani SKP1, which can be induced by oligochitosaccharides, has the following cDNA bases (see Sequence List 1) and protein amino acid sequences (see Sequence List 2).

[0017] The cDNA sequence characteristics of tobacco S-phase kinase-related protein 1 gene (SKP1): genome sequence: 653bp, nu...

Embodiment 2

[0023] 1. Differential display of mRNA and cloning material at the 3′ end

[0024] When the susceptible tobacco variety Nicotiana tabacum var.sam sun NN was cultivated in the greenhouse until the seedlings had 4-5 leaves, the leaves were sprayed with 50mg / L chitosan oligosaccharide, and the leaves were sprayed with double distilled water As a control, samples were taken at 8 hours and 168 hours, respectively.

[0025] 2. Total RNA extraction, quantification and integrity detection

[0026] The dust on the leaf surface was washed with double distilled water, ground in liquid nitrogen, and RNA was extracted with TRIZOL (Gibco BRL) reagent. (1) Quantification: measure the absorbance at 260 and 280nm, and calculate A 260 / A 280 values ​​to estimate the purity of total RNA. Pass A 260 Calculate the amount of total RNA from the value. (2) RNA integrity detection: separate total RNA on 1% formaldehyde denatured agarose gel, if there are two clear bands, and the brightness of la...

Embodiment 3

[0081] 1. Prokaryotic expression of SKP1 protein

[0082] (1) According to the SKP1 cDNA sequence, the primers for the expression protein of the SKP1 gene were synthesized in TaKaRa Company:

[0083] SKP1 Upstream Expression Primer (skp1es)-5'

[0084] CGGCC CATATG TCCTCCTCAAAGATGATC 3′

[0085] SKP1 downstream expression primer (skp1ea)-5'

[0086] GCCGC CTCGAG CTCAAATGCCCAAGCATT 3′

[0087]Referring to the multiple cloning site of pET-23b(+), Nde I and Xhol I restriction sites (underlined parts) were introduced at the 5' ends of primers skp1es and skp1ea, respectively.

[0088] (2) Synthesis of SKP1 cDNA

[0089] The SKP1 cDNA sequence to be expressed was amplified using the SKP1 full-length subcloning plasmid pMD18-SKP1 plasmid as a template, and the PCR amplification reaction was carried out in a 25 μl reaction system:

[0090] PCR reaction system: [stock] 1×r×n(50μl) [final]

[0091] Sterile double distilled water: 18.75μl

[0092] PCR buffer: 10× 2.5μl 1×

[...

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Abstract

The invention relates to S-phase kinase-associated protein 1 (SKP1), specifically to a class of proteins generally existed in eukaryotes (tobacco), which is a core subunit of 3 SCF ubiquitin ligase complex. The protein is related with the antiviral activity of tobacco, has molecular weight of 17527.78 Da and isoelectric point of 4.57 and is positioned in cytoplasm, and the Skp1 structural domain thereof is positioned at 4-105 amino acids. The invention has a distinct guiding role in the application of biological pesticides. The cloning of the gene is valuable for disclosing the signal path of chitooligosaccharides for inducing plant resistance, and becomes an important strategy in the application of oligosaccharide biological pesticides.

Description

technical field [0001] The present invention relates to S-phase kinase-associated protein 1 gene, specifically a tobacco S-phase kinase protein 1 gene sequence derived from the disease-resistant tobacco variety Nicotiana tabacum var. Samsun NN and its encoded protein sequence and apply. Background technique [0002] S-phase kinase-associated protein 1 (S-PHASE KINASE-ASSOCIATED PROTEIN1, SKP1) is a ubiquitous protein in eukaryotes (tobacco), and is the core subunit of the ubiquitin ligase 3SCF complex. In recent years, studies have found that the ubiquitin system mediated by SCF plays a role in biological reactions such as the jasmonic acid signaling pathway and R-gene-mediated resistance, but the specific pathway is not yet clear, especially in chitosan oligosaccharides. The role in the induction of has not been reported yet. Contents of the invention [0003] The purpose of the present invention is to provide a tobacco S-phase kinase protein 1 gene sequence and its enc...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/82A01N65/02A01N65/38
CPCY02A50/30
Inventor 杜昱光张付云白雪芳
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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