Scenedesmus obliquus chloroplast homologous recombination empty vector and application thereof
A technology of Scenedesmus obliquus and homologous recombination, which is 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, improving performance, and increasing protein content
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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 the template, PCR amplification was carried out 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 ...
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 SEQ ID 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 wa...
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