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A ribosome and gene technology, applied in genetic engineering, plant genetic improvement, sugar derivatives, etc.
Inactive Publication Date: 2005-01-19
INST OF BOTANY CHINESE ACAD OF SCI
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However, most of the work is limited to the separation and activity analysis of RIPs proteins by techniques such as chromatography (Vivanco et al., Plant Physiol, 1999 119: 1447-1456; Krawetz et al., Eur JBiochcm, 2000, 267: 1966-1974)
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[0044] Specific embodiments of the present invention: use RA39 gene to obtain ribosome inactivating protein with enzymatic activity
[0045] 1. Acquisition of RA39p protein coding region
[0046] According to SEQ ID NO.1, synthetic gene-specific primer P5: 5'-GAT CACCAGCTTAGCCGTC-3' (nucleotides: 33-49) and P6: 5'-GATC ACACCTTGCATTAATACAACCA-3' (nucleotides: 935-955). An EcoRI restriction site was added to the 5' end of P5, and a SalI restriction site was added to the 5' end of P6 (shown in square brackets). The coding region (ORF) of the gene was amplified by RT-PCR.
[0047] The synthesis conditions of the first-strand cDNA are: 5 μg RNA and 0.5 μg oligo-dT 15 Mix the primers, add sterile water to make the volume of the mixture reach 12 μl, incubate at 70°C for 10 minutes, transfer to an ice bath, add 4 μl 5X first strand buffer, 1 μl 10mM dNTPs, 2ul 0.1mM DTT, preheat at 42°C for 2 minutes , add 1 μl SuperScript II RNaseH - Reverse transcriptase (200U / μl, GIBCO-BRL,...
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Abstract
A ribosome inactivating-protein gene RA39 is cloned out via subtractive hybridization of paddy rice. It can code a ribosome inactivating protein RA39p. The gene related in the present invention can be used to prepare recombinant ribosome inactivating protein for developing biologic preparations and to increase the expression of ribosome protein in plant by genetic technique for improving the disease-resistant power of plant.
Description
technical field [0001] The invention belongs to the field of biotechnology. Background technique [0002] Ribosome-inactivating proteins (RIPs) are a class of plant proteins that are widely distributed in nature, and are distributed in roots, leaves, and seeds of plants (Bass et al., Plant Cell, 1992, 4: 225-234 ; Vivanco et al., Plant Physiol, 1999, 119: 1447-1456; Krawetz et al., Eur J Biochem, 2000, 267: 1966-1974; Van Damme et al., Eur J Biochcm, 267: 2746-2759; Song et al., Plant Mol Biol , 43:439-450). According to the molecular structure characteristics of RIPs, they are divided into three types, namely type I, type II and type III. [0003] Among them, type I RIPs are a class of single-subunit enzymes, such as pokeweed antivirus protein (PAP) and Trichosanthin (TCS) of Trichosanthes kirilowi; type II RIPs are composed of two chains A heterodimer, in which the A chain is equivalent to type I RIPs, and the B chain is a lectin polypeptide, which can bind to specific ...
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