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Chlorella vulgaris chloroplast homologous recombination empty vector and its application

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: 2020-04-17
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 applied to large-scale cultivation; at the same time, in this mutant strain , exogenous genes are regulated by regulatory elements derived from Chlamydomonas reinhardtii, and the expression efficiency is relatively low

Method used

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  • Chlorella vulgaris chloroplast homologous recombination empty vector and its application
  • Chlorella vulgaris chloroplast homologous recombination empty vector and its application
  • Chlorella vulgaris chloroplast homologous recombination empty vector and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of Endogenous Fragments of Chlorella vulgaris Chlorella

[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'-TTCATGTATTCTGTCTATAAAGCTC-3'

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

[0049] Wherein the amplification product of primer SEQ1-for and SEQ1-rev is SEQ ID NO:1; The amplification product of primer SEQ2-for and SEQ2-rev is SEQ ID NO:2; The amplification of primer SEQ3-for and SEQ3-rev Product is SEQ ID NO:3; The amplif...

Embodiment 2

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

[0057] Design and synthesize the following primers:

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

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

[0060] Using the vector pAPEC as a template, the primers aad A-for and aad A-rev was amplified by PCR, and the reaction program was: 94°C for 5 min pre-denaturation; 94°C for 1 min, 56°C for 30 sec, 72°C for 1 min, a total of 35 cycles; 72°C for 5 min. The PCR amplification product is about 906 bp, which is the spectinomycin resistance gene. After the fragment was subjected to agarose gel electrophoresis, the PCR product purified by gel recovery (Tiangen company kit) was connected to the pMD-18T vector (Sigma company) to obtain a gene containing bar The recombinant plasmid pMD- aad a.

[0061] Based on the above products, pMD18T was used as the starting vector to construct the Chlorella vulgaris chloroplast h...

Embodiment 3

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

[0080] Next, two antibacterial peptide genes (GenBank No.6K50_A; GenBank No.AKA60777.2) with antibacterial activity were inserted into the vector and then introduced into Chlorella vulgaris, and the expression of the two foreign genes was detected to detect the expression of the vector 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 SEQI...

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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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Patent Type & Authority Patents(China)
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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