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Novel ribulose diphosphate carboxylase large-subunit gene derived from pavloca viridis

A technology of ribulose diphosphate carboxylase and Pavlova viridis, applied in the fields of genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve the problems of unknown rbcL coding gene, restricting the research of anabolic metabolism and classification status Confirmation and other issues

Inactive Publication Date: 2016-01-27
徐毅
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Problems solved by technology

So far, the rbcL-encoding gene of Pavlova viridis is unknown, which restricts the study of its anabolic metabolism and the confirmation of its taxonomic status

Method used

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  • Novel ribulose diphosphate carboxylase large-subunit gene derived from pavloca viridis
  • Novel ribulose diphosphate carboxylase large-subunit gene derived from pavloca viridis

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Experimental program
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Embodiment Construction

[0019] The isolation process of Pavlova viridis rbcL gene is:

[0020] (1) Obtain the total mRNA of Pavlova viridis using conventional mRNA extraction and separation means in the art, and use primer OligodT to reverse transcribe it into cDNA by conventional means in the art;

[0021] (2) Using cDNA as a template, design primers PrF and PrR according to the conservation of rbcL homologous genes to amplify the gene in the conserved region. The primer sequence is: PrF: 5'-TGTAATTGTAGAGCGTGA-3'; PrR: 5'- CACCTTCTAGCTTACCTACA-3'; PCR reaction conditions: 94 o C3min; 94 o C30s,60 o C30s,72 o C1min, 30 cycles; 72 o C10min; the size of the obtained PCR product is about 540bp;

[0022] (3) The amplification reaction at the 3' end was carried out using cDNA as a template and using the SMARTRACE cDNA amplification kit, and the amplification primers used were: UPM (10×universal primer mix); elo9-3GTATGTCAGGTGTTGACCACAT. The gradient PCR reaction program is: 94oC3min; 94oC30s, 72oC3m...

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Abstract

1,5-ribulose diphosphate carboxylase is a key enzyme for carbon assimilation of a living body, and rbcL is a subunit of the enzyme. Aiming at a chemoautotrophy type microalgae pavloca viridis which can synthesize organisms by using carbon dioxide, the invention discloses a novel rbcL gene by using a combination method of conserved sequence amplification and RACE in a cloning manner. The obtaining of the gene is both beneficial for anabolism of the pavloca viridis and helpful for accurate identification and classification of pavloca viridis.

Description

technical field [0001] The application belongs to the technical field of gene cloning and isolation, and in particular relates to a gene related to the large subunit of ribulose 1,5-diphosphate carboxylase derived from microalgae and its isolation method. Background technique [0002] Ribulose 1,5-bisphosphate carboxylase is widely distributed in autotrophs and is a key enzyme for assimilating and reducing carbon in autotrophs. Ribulose 1,5-bisphosphate carboxylase is composed of large subunit (rbcL) and small subunit (rbcS), among which rbcL changes slowly in evolution, so the gene cloning and sequence analysis of rbcL encoding gene are not only beneficial Studying the synthetic metabolic pathway of this autotroph is also an important means to determine its taxonomic status. Green Pavlova ( Pavlovaviridis ) is a class of chemoautotrophic eukaryotic microalgae, and a class of marine algae with important resource significance and research value. So far, the rbcL coding gen...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/60
Inventor 不公告发明人
Owner 徐毅
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