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Porphyra yezoensis manganese superoxide dismutase and its preparation method

A superoxide and Porphyra zebra technology, applied in the direction of botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of reduced assimilate formation, cell death, photosynthetic rate decline, etc., to solve the problem of promoting Effect

Inactive Publication Date: 2008-06-18
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In plants, reactive oxygen species can cause plant metabolism inactivation, cell death, photosynthesis rate decline, assimilate formation reduction, and even cause serious consequences such as plant quality decline and yield reduction.

Method used

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  • Porphyra yezoensis manganese superoxide dismutase and its preparation method
  • Porphyra yezoensis manganese superoxide dismutase and its preparation method
  • Porphyra yezoensis manganese superoxide dismutase and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0060] Construction of Porphyra zebra cDNA library (using oligonucleotide capping method (Olige-capping), refer to Maruyama et al., 1994):

[0061] (1), extraction of total RNA:

[0062] Total RNA was extracted using UNIQ-10 Column Trizol Total RNA Extraction Kit (purchased from Shanghai Bioengineering Co., Ltd.). The material was obtained from the Porphyra zebra filaments of the algae bank in our laboratory, and the total RNA was extracted by an improved one-step method. Specific steps are as follows:

[0063] Wash with sterile water and blot dry with filter paper, weigh 70 mg of algae, and freeze in liquid nitrogen for 20 minutes. After fully grinding with liquid nitrogen, transfer the algae powder to a pre-cooled 1.5ml EP tube, add 450μl Trizol, vortex vigorously for 30s, and mix thoroughly. with 26-G # The needle was cut twice to form a homogenate. Transfer the homogenate to a new 1.5ml RNase-free EP tube, add 100μl chloroform / isoamyl alcohol, vortex vigorously for 30...

Embodiment 2

[0122] Database construction:

[0123] Porphyra zebra cDNA library constructed by oligonucleotide capping method (Olige-capping) was used as the library source for sequencing EST. The DNA sequencing work was completed by Hangzhou Huada Genome Research Center. The universal primer used is M13F (5'-GTAAAACGACGGCCAGT-3'), and the automatic sequencer is MegaBace TM 1000.

[0124] After removing the carrier sequence in the sequence and correcting the ambiguous bases detected at the 3' end, the sequence with a fragment length of less than 100 is regarded as an invalid sequence, and the rest of the sequence is processed using the EST Analysis pipeline System TM Assist with bioinformatics analysis to determine function and reduce redundancy. The program combines common tools for bioinformatics analysis, including BLAST, Phred, Pfam, Crossmatch, Phrap, Cap3, etc. A total of 469 EST sequences representing different genes were obtained, and the non-redundant genes were searched for h...

Embodiment 3

[0126] Select the required strain, save, sequence and similarity analysis:

[0127]According to the results of EST analysis, the Mn-SOD genes related to the stress resistance of Porphyra were selected. The host cell of the strain is human enterobacteria competent cell DH5α, and the plasmid vector is pMD18-T. The strain preservation number is CGMCC No.1414.

[0128] The sequence of the inserted fragment of the strain was determined by Shanghai Yingjun Biotechnology Co., Ltd. Most of the sequence was determined by bidirectional sequencing, and a 958bp nucleotide sequence was obtained, which is the cDNA of the Mn-SOD gene of Porphyra zebra sequence. The sequence has been submitted to the International Gene Sequence Bank (GenBank) with the sequence number DQ146477. The open reading frame of the sequence is 675bp (ATG-TAA), can translate 224 amino acid residues and stop codon UAA, and the predicted molecular weight is 24469.09Da (application analysis software DNAsis). See SEQ N...

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Abstract

The invention relates to a Porphyra yezoensis superoxide dismutase with preparation method, which comprises 224 amino residues with predictive molecular weight as 24469.09Da. Wherein, the most of 24 residues on N-end are hydrophobic amino acid, this peptide segment plays an role in transfering the enzyme to mitochondria; the histidine (H) on 27th, 84th, 178th sites of the sequence, and aspartic acid (D) on 174th site, are all the combination sites with Mn2+; there is '' DVWEHAYYL '' as conservative metal combination structure domain; compared with the sequence of Mn-SOD amino acid, this enzyme has 50-60% isogeny. This invention uses enzyme cDNAsequence and expression carrier pSE380 to construct recombinant plasmid pSE380 / MnSOD and express int escherichia coli DH5alpha; the method is precise and reasonable and exploits the material resource for superoxide dismutase.

Description

technical field [0001] The invention relates to the improvement of the bioengineering technology of aquatic algae, in particular to a manganese superoxide dismutase (Mn-SOD) of Porphyra variegata and a preparation method thereof. It includes the construction of cDNA library in Porphyra zebra, the cloning of Mn-SOD, the expression in Escherichia coli, the research of bioengineering technology such as product purification and activity detection. Background technique [0002] Porphyra yezoensis belongs to Rhodophyta and is the main cultivated species of laver in northern China. Now, there are still some problems that need to be solved urgently in the large-scale cultivation of laver, such as controllable filamentous growth and fast growth of thallus, high yield, high quality, disease resistance, stress resistance, anti-algae epiphysis, anti-rot, etc. . In order to solve the stress resistance problem of laver, Feng Chen and others have studied salinity stress, Cu 2+ According...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/08C12N1/21C12N15/70C12Q1/26
Inventor 茅云翔隋正红王荣周晓君王孟强
Owner OCEAN UNIV OF CHINA
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