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A kind of homologous recombination empty vector of Scenedesmus obliquus chloroplast and its application

A technology of Scenedesmus obliquus and homologous recombination, applied in the field of genetic engineering, can solve problems that hinder basic research and application development, and achieve the effects of increasing immune performance, increasing protein content, and efficient transcription

Active Publication Date: 2020-04-28
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on the chloroplast transformation system of Scenedesmus obliquus, which seriously hinders the progress of basic research and application development of this algae.

Method used

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  • A kind of homologous recombination empty vector of Scenedesmus obliquus chloroplast and its application
  • A kind of homologous recombination empty vector of Scenedesmus obliquus chloroplast and its application
  • A kind of homologous recombination empty vector of Scenedesmus obliquus chloroplast and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Cloning of chloroplast endogenous fragments of Scenedesmus obliquus

[0034] Design and synthesize the following primers:

[0035] SEQ1-for: 5'-TGCTCGCAAGAGTGAAAACTCAAAG-3'

[0036] SEQ1-rev: 5'-TTAAAAAAGCTGGACCATACTGGACTTG-3'

[0037] SEQ2-for: 5'-AGGTAAAAAAGGGAATATAGCTC-3'

[0038] SEQ2-rev: 5'-TCGCCGGCTCATTCTTCAACAG-3'

[0039] SEQ3-for: 5'-ATAAAAATTTTAAGTTTCAAATTTTTA-3'

[0040] SEQ3-rev: 5'-AATATAAAAAAATAAAAATTTAAAATTCTCC-3'

[0041] SEQ4-for: 5'-ATCAAAAAAAATGTTTTTTTTTGA-3'

[0042] SEQ4-rev: 5'-ATAAAAAATAAAAAAAGTATT-3'

[0043] SEQ5-for: 5'-TTTTTTTTTAAAATACTTCCTCTTTAAAG-3'

[0044] SEQ5-rev: 5'-CAGGTTCTTCCCAAAATTGCG-3'

[0045]The amplification product of primers SEQ1-for and SEQ1-rev is SEQ ID NO:1; the amplification product of primers SEQ2-for and SEQ2-rev is SEQ ID NO:2; the amplification of primers SEQ3-for and SEQ3-rev The product is SEQ ID NO:3; the amplification product of primers SEQ4-for and SEQ4-rev is SEQ ID NO:4; the amplification pr...

Embodiment 2

[0051] Example 2: Construction of an empty vector for chloroplast transformation of Scenedesmus obliquus

[0052] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’

[0053] bar-rev: 5’-TCATCAAATCTCGGTGACGGG-3’

[0054] Using the vector pSVB as a template, PCR amplification was performed with primers bar-for and bar-rev. The reaction program was: 94°C for 5 min pre-denaturation; 94°C for 1 min, 54°C for 30 sec, 72°C for 40 sec, a total of 35 cycles; 5min extension at 72°C. The PCR amplification product is about 555 bp, which is the herbicide resistance gene bar. After the fragment was electrophoresed on agarose gel, the gel was recovered (Tiangen company kit) and purified for use.

[0055] Based on the above products, pMD18T was used as the starting vector to construct an empty vector for homologous recombination of the chloroplast of Scenedesmus obliquus by the method of homologous recombination. Wherein pMD-SEQ2, pMD-SEQ3, pMD-SEQ5 need to utilize PCR to add linker and restriction enzym...

Embodiment 3

[0071] Example 3 Application of the vectors obtained according to the above examples in chloroplast transformation of Scenedesmus obliquus

[0072] 1. Construction of expression vector

[0073] Design and synthesize the following primers:

[0074] F1-for: 5’- CCTCTAGA ATG CATCATCACCCATCACC ATGGTTTCGGTTGCAACGGTCCCTGG-3’

[0075] F1-rev: 5’- TTAGTAGCACTTGCAGACGAA TAAATTTT GAAATGGTGATGGTGATGGTGCAT-3’

[0076] F2-for: 5’-ATG CACCATCACCATCACCAT TTTCTTCTTCCACATCATCAAGGG-3’

[0077] F2-rev: 5’- GGGATCC TTACTTCCAGACGAGACCGTGGAT-3’

[0078] Among them, F1-for carries Xba I restriction site and 6×His tag, the underlined sequence fragment of F1-rev is SEQID NO:6, and the italicized part is the linker; the underlined sequence in F2-for is 6×His tag, which is carried by F2-rev Bam HI restriction site.

[0079] The synthetic antimicrobial peptide gene 1 was used as the template, and PCR amplification was carried out with primers F1-for and F1-rev. The reaction program was...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a scenedesmus obliquus chloroplast homologous recombination empty vector and application thereof. The scenedesmusobliquus chloroplast homologous recombination empty vector includes an upstream homologous arm, a downstream homologous arm, a promoter and a terminator, the promoter and the terminator are arranged between the upstream homologous arm and the downstream homologous arm, and a base sequence is inserted between the promoter and the terminator, wherein the base sequence is shown in SEQ ID NO:6, and the base sequence and at least one exogenous gene form a polycistron structure, the upstream homologous arm contains a base sequence shown in SEQ ID NO:1, and the downstream homologous arm contains a base sequence shown in SEQ ID NO:2. By adopting the scenedesmus obliquus chloroplast homologous recombination empty vector, several exogenous genes can be stably expressed in chloroplast.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an empty vector for homologous recombination of chloroplasts of Scenedesmus obliquus and its application. Background technique [0002] Scenedesmus obliquus ( Scenedesmus obliquus ) is a freshwater unicellular eukaryotic green algae, belonging to the Chlorophyta, Chlorophyta, Chlorococcus order Scenedesmus, and usually consists of 2 or 4 cells in a definitive population. The algal cells are spindle-shaped or elliptical, with tapered ends; the free surfaces of the outer cells of the colony are sometimes concave, sometimes raised; the cell wall is flat; the cells are 3-6 µm wide and 13-16 µm long; Population width is 12-24 µm. Cells contain a nuclear protein and a periphyton. The main photosynthetic pigments are chlorophyll a, chlorophyll b, lutein and carotenoids. Under normal conditions, the algal cells are grass-green and reproduce in a nutrient-rich still water ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12R1/89
CPCC12N15/8213C12N15/8214
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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