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cDNA sequence for encoding undaria pinnatifida zeta-carotene dehydrogenase and amino acid sequence and application thereof

A technology of carotene and wakame, applied in the field of genetic engineering, can solve the problem that the biosynthesis pathway of carotenoids is not completely clear

Active Publication Date: 2018-12-21
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The biosynthetic pathway of carotenoids in red and green algae has been fully described, in contrast, in brown algae, the biosynthetic pathway of carotenoids is not fully understood

Method used

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  • cDNA sequence for encoding undaria pinnatifida zeta-carotene dehydrogenase and amino acid sequence and application thereof
  • cDNA sequence for encoding undaria pinnatifida zeta-carotene dehydrogenase and amino acid sequence and application thereof
  • cDNA sequence for encoding undaria pinnatifida zeta-carotene dehydrogenase and amino acid sequence and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Schematic diagram of extracting full-length ZDS gene from Wakame as follows figure 1 shown. figure 1 Among them, A is the electropherogram of the cDNA partial sequence clone of UpZDS gene; B is the electropherogram of the 5'-RACE amplification product (M: 5K DNA Marker; 1: the product of the first round of amplification at the 5' end of ZDS; 2: the product of the 5' end of ZDS The product of the second round of amplification at the end of the ZDS); C is the electropherogram of the 3'-RACE amplification product (M: 5K DNA Marker; 1: the product of the first round of amplification at the 3' end of ZDS; 2: the second round of amplification at the 3' end of ZDS Amplified product); D is the electrophoresis image of the cDNA full-length amplification product of UPZDS gene; E is a schematic diagram of cloning the full-length ZDS gene from Wakame, and indicates the length of the PCR amplified fragment and the PCR primers used.

[0043] The operation steps are as follows:

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Embodiment 2

[0076] Embodiment 2, wakame -carotene desaturase gene sequence biological information analysis and protein structure prediction

[0077] The sequencing results of the three sequences were spliced, and the full-length cDNA sequence of the ZDS gene of Wakame was deduced. Then, the full-length cDNA sequence was translated into a protein sequence using the bioinformatics software DNAMAN (LynnonBiosoft, USA). On this basis, the deduced amino acid sequence of Undaria pinnatifida Suringar ZDS was compared with that of Ectocarpus siliculosus ZDS (CBN76924.1), Nannochloropsis gaditana ZDS (EWM29966.1), Chrysochromulina sp. Multiple sequence alignment of ZDS (KOO28349.1), Phaeodactylum tricornutum ZDS (XP_002176685.1), and Fistulifera solaris ZDS (GAX15703.1) were performed to compare the importance of Wakame ZDS at the amino acid level. Differences between functional loci and other closely related algae.

[0078]In addition, use ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) ...

Embodiment 3

[0084] Example 3, construction and identification of expression vector pET-15b-ZDS

[0085] The PCR-amplified UpZD gene sequence was double-digested with BaMHI and XHOI restriction enzymes, cloned with primers ZDSCF and ZDSCR primers, purified and connected to the PET-28a(+) vector to form a recombinant plasmid pET-28a-UPZDS , carrying a 6×His tag at the N-terminus.

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Abstract

The invention relates to cDNA sequence for encoding undaria pinnatifida zeta-carotene dehydrogenase and the amino acid sequence and application thereof. The full-length cDNA sequence of the undaria pinnatifida zeta-carotene dehydrogenase gene is separated and obtained from undaria pinnatifida through a means of rapidly amplification of cDNA teiminal and named UPZDS, the full-length cDNA sequence is 2804bp, the open reading frame (ORF) containing 1821bp encodes 606 amino acids, and the molecular weight is 65.82717kDa. Cloning of the undaria pinnatifida zeta-carotene dehydrogenase (ZDS) gene lays basis for researching biosynthetic pathway of fucoxanthin in brown seaweed, and has the important meaning on medicine and health care and industrialized production of carotenoid in brown seaweed.

Description

Technical field: [0001] The invention relates to a coding wakame - Cloning and functional validation of the carotene dehydrogenase (ZDS) gene, especially involving a gene encoding Wakame - The cDNA sequence of carotene dehydrogenase and its amino acid sequence and application belong to the technical field of genetic engineering. Background technique: [0002] Carotenoids are divided into two categories: one is the oxygen-free hydrocarbons, that is, carotene; the other is the oxygen-containing derivatives of carotene, that is, lutein. Fucoxanthin, also known as fucoxanthin, belongs to the xanthophyll class of carotenoids and is a type of fat-soluble pigment widely distributed in Phaeophyta and Diatoms. The biosynthetic pathway of carotenoids in red and green algae has been fully described, in contrast, in brown algae, the biosynthetic pathway of carotenoids is not fully understood. Therefore, studying the genes of key enzymes involved in the carotenoid biosynthetic pathw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02
CPCC12N9/001C12Y103/99026
Inventor 李荣贵马怡杜桂彩张廷婷
Owner QINGDAO UNIV