A kind of lipid-rich Nannochloropsis chloroplast transgenic system and its application
A technology of Nannochloropsis and chloroplasts, which is applied in the field of genetic engineering, can solve the problems affecting the expansion of mutant strains, hindering the progress of basic research and application development, and the lack of lipid-rich Nannochloropsis chloroplast transformation technology. Effects of photosynthetic growth rate, highly efficient endogenous regulatory sequences, and stable insertion
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Embodiment 1
[0036] Example 1 Cloning of endogenous fragments of chloroplasts
[0037] Design and synthesize the following primers:
[0038] SEQ1-for: 5’-CTAGGAGACTTGAGTATAGTAGG-3’
[0039] SEQ1-rev: 5’-CTGGGCTATCCTGGACTTGAACCA-3’
[0040] SEQ2-for: 5’-TGGGGGTATAGCTCAGCTGGTAGAG-3’
[0041] SEQ2-rev: 5’-TCGGGGAGAACCAGCTAGCTCCGG-3’
[0042] SEQ3-for: 5’-ATTTTTGTTGTTGACTAAAACAC-3’
[0043] SEQ3-rev: 5’-AATGAGTACTAGAATAAATAGGG-3’
[0044] SEQ4-for: 5’-GAACATAAAAGAGGTTTTAAAATTACT-3’
[0045] SEQ4-rev: 5’-TATAGCAGTAAATTCTCTGGTTCTCG-3’
[0046] SEQ5-for: 5’-ATAATACATAACAACTAAAACCAA-3’
[0047] SEQ5-rev: 5’-GTACAATGTGTTAGGTCTTACAAAT-3’
[0048] SEQ6-for: 5’-GAATATTTAATTACATATGAGTGA-3’
[0049] SEQ6-rev: 5’-ATTAAATTAACAAAGAAAATCTG-3’
[0050] 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 product of primers SEQ3-for and SEQ3-rev The product is SEQ ID NO: 3; the amplification product of primers SE...
Embodiment 2
[0057] Example 2: Construction of empty vector for chloroplast transformation of Pseudochlorococcum lipid-rich
[0058] Design and synthesize the following primers:
[0059] bar-for: 5’-ATGAGCCCAGAACGACGCC-3’
[0060] bar-rev: 5’-TCATCAAATCTCGGTGACGGG-3’
[0061] Using the vector pSVB as a template, PCR amplification was carried out with primers bar-for and bar-rev. The reaction program was: 94℃ 5min pre-denaturation; 94℃ 1 min, 54℃ 30 sec, 72℃ 40 sec, a total of 35 cycles; Extension at 72°C for 5 min. The PCR amplification product is about 555 bp, which is the herbicide resistance gene bar . 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- bar .
[0062] Based on the above products, pMD18T was used as the starting vector to construct a homologous recombination vector for the chloroplast of Pseudochlorococcum lipid-rich microalgae thro...
Embodiment 3
[0079] Example 3 Application of the vector obtained according to the above example in the transformation of chloroplasts of Pseudochlorococcum lipo-rich
[0080] Next, insert the two antibacterial peptides (GenBank No.6K50_A; GenBank No.AKA60777.2) genes with antibacterial activity into the vector and then introduce them into the microsphaeropsis lipid-rich, and detect the expression of these two foreign genes. The performance of the carrier.
[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:
[0085] TGGTGATGGTGATGGTGCAT AAAATATCTACTC TTAGTAGCACTTGCAGACGAA
[0086] F2-for: 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 SEQID NO: 7, the underlined sequence i...
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