A Dunaliella salina chloroplast homologous recombination empty vector and its application

A Dunaliella salina, homologous recombination technology, applied in the field of genetic engineering, can solve the problems of restricting basic research and application development, lack of high-efficiency and stable endogenous high-efficiency regulatory sequences at chloroplast insertion sites, and achieve high-efficiency endogenous regulatory sequences Effect

Active Publication Date: 2019-08-23
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

At present, the whole genome sequence of the chloroplast genome of Dunaliella salina has been carried out, which provides a sufficient basis for the genetic modification of Dunaliella salina chloroplast, but so far, the research on the transformation of Dunaliella salina chloroplast has just started, and there is a lack of efficient and stable chloroplast insertion sites and Endogenous high-efficiency regulatory sequences restrict the basic research and application development of this algae

Method used

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  • A Dunaliella salina chloroplast homologous recombination empty vector and its application
  • A Dunaliella salina chloroplast homologous recombination empty vector and its application
  • A Dunaliella salina chloroplast homologous recombination empty vector and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cloning of Dunaliella salina chloroplast homologous recombination fragment

[0034] Design and synthesize the following two pairs of primers:

[0035] P1: 5'-TTACCAGGGTTTGACATGTCTAGAA-3'

[0036] P2: 5'-TGGGCTATAGAAGATTTGAAC-3'

[0037] P3: 5'-GGGAATGTAGCTCAGTTGGTAGAGC-3'

[0038] P4: 5'-TTCAGCTGTTTCGTTTTTAGAAAACT-3'

[0039] Wherein the amplified product of primers P1 and P2 is SEQ ID NO: 1, i.e. fragment 16S-TrnA; the amplified product of primers P3 and P4 is SEQ ID NO: 2, i.e. fragment TrnI-23S (see figure 1 ).

[0040] Using the total genome DNA of Dunaliella salina as a template, carry out PCR amplification with primers P1 and P2, the reaction program is: 94°C 10min pre-denaturation; 94°C 1min, 60°C 90sec, 72°C 90sec, a total of 30 cycles; 72°C 5min extend. The PCR amplification product is about 901bp, which is the fragment 16S-trnI. After the fragment is subjected to agarose gel electrophoresis, the PCR product purified by gel recovery (Tiangen Co...

Embodiment 2

[0042] Example 2 Amplification and cloning of two chloroplast promoter fragments of Dunaliella salina

[0043] Design and synthesize the following two pairs of primers:

[0044] P5: 5'-ATCCGCGTAGAGTAATAGG-3'

[0045] P6: 5'-GAGCACCATTTTTACTTCTGGTGTA-3'

[0046] P7: 5'-GGATCCGCCGATCCGTGGTTTAGAGTT-3'

[0047] P8: 5'-ACGTGCCCAAAGGCTAGTATTT-3'

[0048] Wherein the amplified product of primers P5 and P6 is SEQ ID NO: 3, that is, fragment 5'atpA, which is a promoter with chloroplast activation function derived from Dunaliella salina chloroplast; the amplified product of primers P7 and P8 is SEQ ID NO : 4, namely the fragment 5' psbA, is derived from the promoter with chloroplast activation function of Dunaliella salina chloroplast (see figure 2 ).

[0049] Using the total genome DNA of Dunaliella salina as a template, PCR amplification was carried out with primers P5 and P6. The reaction program was: 94°C 10min pre-denaturation; 94°C 1min, 60°C 90sec, 72°C 90sec, a total of 30 c...

Embodiment 3

[0051] Example 3 Construction of Dunaliella salina chloroplast homologous recombination empty vector

[0052] Based on the above-mentioned cloning vectors pMD16I, pMD23A, pMDatpA, and pMDpsbA, a Dunaliella salina chloroplast homologous recombination vector was constructed by means of homologous recombination.

[0053] Design and synthesize the following six pairs of primers:

[0054] P9: tagcctttgggcacgtATGAGCCCAGAACGACGCC

[0055] P10: ctgagctacattcccTCATCAAATCTCGGTGACGGG

[0056] P15: tgctcctcgagCTGCTTGTGAAGTTTGGAAAGAAA

[0057] P16: tgctcctcgagCTGCTTGTGAAGTTTGGAAAGAAA

[0058] P17: catgattacgaattcggatccTTACCAGGGTTTGACATGTCTAGAA

[0059] P18: gcggatTGGGCTATAGAAGATTTGAAC

[0060] P19: gatgaGGGAATGTAGCTCAGTTGGTAGAGC

[0061] P20: acgacggccagtgccaagcttTTCAGCTGTTTCGTTTTTAGAAAACT

[0062] P21: tatagcccaATCCGCGTAGAGTAATAGG

[0063] P22: aagcagctcgagGAGCACCATTTTTACTTCTGGTGTA

[0064] P23: tatgaccatgattacgaattcGGATCCGCCGATCCGTGGTTTAGAGTT

[0065] P24: tcatACGTGCCCAAAGGCTAG...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a Dunaliella salina chloroplast homologous recombination empty vector and its application. The vector includes a promoter and a terminator, and the recombinant empty vector contains the upstream homology arm of the base sequence shown in SEQ ID NO: 1 and the downstream homology arm of the base sequence shown in SEQ ID NO: 2, the homology arm The base sequence shown in SEQ ID NO: 5 is inserted with at least one foreign gene to form a polycistronic structure. The Dunaliella salina chloroplast stable expression system of the present invention can realize the stable expression of multiple exogenous genes in the chloroplast.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a Dunaliella salina chloroplast homologous recombination empty vector and its application. Background technique [0002] In photosynthetic eukaryotic microalgae, the nuclei, chloroplasts, and mitochondria all contain DNA, which constitutes an independent but interrelated genetic system. Since the birth of genetic engineering technology, the technology of exogenous gene transformation targeting the nucleus has been widely used. However, with the progress of research, people found that nuclear genome genetic engineering has insurmountable difficulties: 1. The efficiency of exogenous gene insertion into nuclear genome is low, and the insertion is random, resulting in great variability among different clones, so extensive research is required. 2. The structure and function of the nuclear genome are complex, and the insertion of foreign genes may sometimes cause variation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
CPCC12N15/8213C12N15/8214C12P23/00C12N1/125C12R2001/89
Inventor 崔玉琳林彬秦松初金玲焦绪栋
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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