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A technology of spinosa and nucleic acid molecules, which is applied in the field of genetic engineering and can solve problems such as the difficulty of amplifying the target gene sequence
Active Publication Date: 2021-04-13
SHENZHEN UNIV
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The advantage of this technique is that the amplification of a gene with a known sequence is the most convenient method of gene cloning, that is, the gene sequence reported by others is directly used as the basis for its own cloning. It has not been reported yet. Without accurate reference sequences and PCR amplification methods, it is particularly difficult to amplify the target gene sequence on Biscellum algae. It is not only necessary to search for the nuclear proliferation antigen gene sequence (pcna ) as a basis, it is necessary to explore the method of amplifying and cloning the gene
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Embodiment 1
[0038] Example 1 Obtaining of Cochlodinium geminatum Nuclear Proliferation AntigenGene
[0039] 1 Extraction of total RNA from Cochlodinium geminatum (this process was performed on ice)
[0040] ① Take 50 mL of Cochlodinium geminatum and let it stand in a centrifuge tube. After the algae liquid naturally settles at the bottom, centrifuge at 8000 rpm for 5 minutes. After absorbing the supernatant, quickly add 500 μL of RNAiso Plus (TAKARA, product number 9108);
[0041] ② Add 100 μL of chloroform, turn it upside down 5-6 times, and let it stand at room temperature for 10 minutes;
[0043] ④Take 200 μL of the supernatant, add 200 μL of isopropanol (pre-cooled at -20°C), invert up and down 5-6 times, and place at room temperature for 10 minutes;
[0044] ⑤ 4°C, 10000rpm, centrifuge for 15min;
[0045] ⑥ Discard the supernatant, add 500 μL 75% (v / v) ethanol (3 mL ethanol + 1 mL RNase free water) to wash, 4 ° C, 10000 rpm, centrif...
Embodiment 2
[0075] Example 2 Changes in the expression level of Spirulina pcna gene under the stress of alginolytic bacteria
[0076] 1. Add the supernatant of algae-lytic bacteria to the twin-celled Spirulina cultured in 2216E liquid medium, so that the volume percentage of the supernatant of algae-lytic bacteria in the liquid medium is 3%, which is the treatment group; add An equal amount of 2216E culture medium was used as a control group, and 100 mL of algae liquid of the control group and the treatment group were taken at 7 h and 20 h respectively, and RNA was extracted (the specific method was the same as in Example 1). The process of obtaining the supernatant of algae-lytic bacteria is as follows: 1ml of the bacterial solution is added to 100mL of 2216E liquid medium, cultured at 30°C and 180rpm for 48h, centrifuged at 12000rpm for 10min, and the supernatant is filtered through a 0.22μm filter membrane. have to.
[0077] 2. Extracted total RNA, using PrimeScript TM RT reagent Kit...
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Abstract
The invention relates to the field of geneengineering, in particular to a cochlodinium geminatum proliferating cell nuclear antigen (pcna) gene and application thereof. The amplification length of the pcna gene is 430bp, and the nucleotide sequence of the pcna gene is SEQ NO.1. In the aspect of application, an amino acid sequence (SEQ ID NO: 2) phylogenetic tree of the gene is constructed, evidences in the aspect of protein can be provided for researching phylogenetic development of dinoflagellate, the expression quantity change of the gene under the stress of algicidal bacteria supernatant is explored, a foundation is laid for researching abiotic stress response, and gene sources and technical support are provided for exploring the death mechanism of marine algae.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a gene of a spinosa dicystis cellnucleus proliferation antigen and an application thereof. Background technique [0002] The most commonly used method for cloning the target gene from algae is the PCR amplification cloning method, that is, a method for cloning the gene sequence on the basis of the target gene sequence of the known algae strain. First find the relevant gene sequence from the gene library (GenBank), synthesize oligonucleotide primers accordingly, extract DNA or RNA from the organism, perform PCR amplification, and insert the amplified target fragment into a suitable plasmid vector after purification Above, the recombinants were detected by restriction analysis and sequence analysis, and compared with known gene sequences to obtain the target gene. The advantage of this technique is that the amplification of a gene with a known sequence is the most convenient me...
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