Chlorella vulgaris chloroplast homologous recombinant empty vector and application thereof

A technology of homologous recombination in Chlorella vulgaris, applied in the field of homologous recombination empty vectors in Chlorella vulgaris chloroplasts, can solve the problems of low efficiency and inability to apply large-scale cultivation, and achieve rapid growth, improved expression, and high nutritional value Effect

Active Publication Date: 2020-01-10
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this vector is based on the mutation of a functional gene. Photosynthesis is not affected, but the dark reaction is affected. The mutant strain of Chlorella obtained after transformation cannot be applie

Method used

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  • Chlorella vulgaris chloroplast homologous recombinant empty vector and application thereof
  • Chlorella vulgaris chloroplast homologous recombinant empty vector and application thereof
  • Chlorella vulgaris chloroplast homologous recombinant empty vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0035] Example 1 Cloning of endogenous fragments of Chlorella vulgaris chloroplast

[0036] Design and synthesize the following primers:

[0037] SEQ1-for: 5’- caaacattga acctagaat-3’

[0038] SEQ1-rev: 5’-CACCATGGTGGGCTATGCTGG-3’

[0039] SEQ2-for: 5’-TTCCATTTTTGGGGAGAAAAGG-3’

[0040] SEQ2-rev: 5’-TCACCCAAGTTTCATCCTGGT-3’

[0041] SEQ3-for: 5’-CCGGAAATTGATACAATAGATTTCA-3’

[0042] SEQ3-rev: 5’-TCCGTGAAAACTTTTGTTTTTCACA-3’

[0043] SEQ4-for: 5’-ATGAATCTCTTGGAACTATGGTATCA-3’

[0044] SEQ4-rev: 5’- TCTAATAAAAAGTGTATGGTTTACGAT-3’

[0045] SEQ5-for: 5’-TTTTTTTTCCTCTCTTAAGCTTCTTAGC-3’

[0046] SEQ5-rev: 5’-CAGGTCTCTTCCCAAAATTGCG-3’

[0047] SEQ6-for: 5’-TTCATGTATTCTGTCTATAAAGCTCTC-3’

[0048] SEQ6-rev: 5’-GAGTACTTGAGAAATAAAGAGG-3’

[0049] The amplification products of primers SEQ1-for and SEQ1-rev are SEQ ID NO:1; the amplification products of primers SEQ2-for and SEQ2-rev are SEQ ID NO: 2; the amplification products of primers SEQ3-for and SEQ3-rev The product is SEQ ID NO: 3; the amplification p...

Example Embodiment

[0056] Example 2: Construction of an empty vector for chloroplast transformation of Chlorella vulgaris

[0057] Design and synthesize the following primers:

[0058] aad A-for: 5’-ATGCCTCGGGCATCCAAGCAGCA-3’

[0059] aad A-rev: 5’-TTATTTGCCGACTACCTTGGTGATC-3’

[0060] Using vector pAPEC as template, primer aad A-for and aad A-rev carries out PCR amplification, the reaction program is: 94℃ 5min pre-denaturation; 94℃ 1 min, 56℃ 30 sec, 72℃ 1 min, 35 cycles; 72℃ 5min extension. The PCR amplified product is about 906 bp, which is the spectinomycin resistance gene. After the fragment was electrophoresed on agarose gel, the PCR product purified by gel recovery (Tiangen kit) was connected with pMD-18T vector (Sigma) to obtain the gene bar Recombinant plasmid pMD- aad A.

[0061] Based on the above products, pMD18T was used as the starting vector to construct the Chlorella vulgaris chloroplast homologous recombination vector through the homologous recombination method. Among them, pMD-S...

Example Embodiment

[0079] Example 3 Application of the vector obtained according to the above example in the transformation of Chlorella vulgaris chloroplast

[0080] Next, insert two antimicrobial peptide genes (GenBank No.6K50_A; GenBank No.AKA60777.2) with antibacterial activity into the vector and then introduce them into Chlorella vulgaris, and detect the expression of these two foreign genes to detect the vector’s performance.

[0081] 1. Construction of expression vector

[0082] Design and synthesize the following primers:

[0083] F1-for: 5’- CCTCTAGA ATG CATCATCACCATCACCAT GGTTTCGGTTGCAACGGTCCCTGG-3’

[0084] F1-rev:5’- TGGTGATGGTGATGGTGCAT ATTCTAGGTTCAATGTTTG TTAGTAGCACTTGCAGACGAA-3’

[0085] F2-for:

[0086] 5’-ATG CACCATCACCATCACCAT TTCTTCTTCCACATCATCAAGGG-3’

[0087] F2-rev: 5’- GGGATCC TTACTTCCAGACGAGACCGTGGAT-3’

[0088] Among them, F1-for carries Xba I restriction site and 6×His tag, the underlined sequence fragment of F1-rev is SEQ IDNO: 7, the underlined sequence in F2-for is 6×His ...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a chlorella vulgaris chloroplast homologous recombinant empty vector and application thereof. The recombinant empty vector comprises upstream and downstream homologous arms, at least one promoter and at least one terminator are arranged between the upstream and downstream homologous arms, and a base sequence which is as shown in SEQ ID NO: 7 and forms a polycistron structure with at least one exogenous gene is inserted between the promoter and the terminator; and the upstream homologous arm contains a base sequence shown as SEQ ID NO: 1, and the downstream homologous arm contains a base sequence shown as SEQ ID NO: 2. By adopting a chlorella vulgaris chloroplast stable expression system disclosed by the invention, stable expression of a plurality of exogenous genes in chloroplast can be realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a chloroplast homologous recombination empty vector of Chlorella vulgaris and its application. Background technique [0002] Chlorella is a class of unicellular microalgae belonging to Chlorphyta, Chlorophyceae, Chlorococcales, ChlorellaAceae, and Chlorella. At present, there are about 10 species of chlorella known in the world, the common ones are Chlorella vulgaris, Chlorella zofingiensis, Chlorella ellipsoidea and Chlorella protothecoides )Wait. [0003] Chlorella is a single-celled green algae that is spherical or oval in shape, 3-12 µm in diameter, with single swimming cells or clusters. Chlorella is widely distributed in nature and has a fast growth and reproduction speed. It is the only animal and plant on the earth that can grow four times in 20 hours, and can obtain higher cell density and biomass; and Chlorella is easy to cultivate and can use light. Ener...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N1/13C12R1/89
CPCC12N15/65C12N15/8213
Inventor 崔玉琳王康任家利秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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