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Method for constructing chlorella expression vector, converting chlorella and breaking wall of chlorella

An expression vector, chlorella technology, applied in the field of plant genetic engineering, to achieve the effect of reducing costs and steps, increasing wall breaking rate, and increasing yield

Active Publication Date: 2009-06-17
北京中加保罗生物科技有限公司
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  • Abstract
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Problems solved by technology

[0009] In this study, the nitrate reductase gene deletion mutant of Chlorella ellipsoides was used as the recipient, NPTII and nitrate reductase gene were used as screening markers, and Ubiquitin promoter was used to control NP -1, adopt the economical and practical Chlorella PEG transformation method to transform, use NPTII and nitrate reductase gene as double screening markers in the initial screening, carry out double screening pressure screening with G418 and nitrate (3 solid culture plate screening and 3 liquid culture screenings), and then screened only with nitrate, and obtained transgenic Chlorella that can be cultivated on nitrate medium and has strong NP-1 activity. It has not been reported that removing the G418 screening pressure can NP-1 transgenic strain with strong NP-1 activity

Method used

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  • Method for constructing chlorella expression vector, converting chlorella and breaking wall of chlorella
  • Method for constructing chlorella expression vector, converting chlorella and breaking wall of chlorella
  • Method for constructing chlorella expression vector, converting chlorella and breaking wall of chlorella

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Embodiment 1

[0041] Embodiment 1. Expression vector construction method of Chlorella ellipsoides nitrate reductase mutant

[0042] Primers were designed according to the Ubiquitin promoter sequence (SEQ ID NO: 1), upstream primer: 5'CCGGAAGCTTGTGCAGCGTGACCCG3' (SEQ ID NO: 2); downstream primer: 5'GCCCGGATCCCTGCAGAAGT3' (SEQ ID NO: 3), wherein in the upstream primer A Hind III restriction site was added, a BamHI restriction site was added to the downstream primers, and the Ubiquitin promoter was obtained from the maize genome by PCR. The sequencing results showed the Ubiquitin promoter sequence (SEQ ID NO: 4, which is in SEQ ID NO; A Hind III restriction site and a BamH I restriction site were added to the 5' end and 3' end of 1, respectively). The PCR product was double-digested with HindIII and BamHI endonucleases, plasmid pBI221 (Clontech) ( figure 1 ) was also digested with Hind III and BamH I endonucleases at 16°C overnight, and the ligation product was transformed into Escherichia co...

Embodiment 2

[0045] Example 2. Using the pGreen0029 framework to construct an expression vector.

[0046] Primers were designed according to the Nos terminator sequence (SEQ ID NO: 7), upstream primer: 5'ATAAGAATGCGGCCGCTCGAATTTCCCCGATCGTTCAAAC3' (SEQ ID NO: 8) downstream primer: 5'CGAGCTCGCCCGATCTAGTAACATAGATGA3' (SEQ ID NO: 9), where Not was added to the upstream primer I enzyme cutting site, add the Sac I enzyme cutting site in the downstream primer, and obtain the Nos terminator from pBI221 (Clontech) by PCR method. The PCR product is double-digested with NotI and Sac I endonucleases, and the plasmid pGreen0029 (BBSRC)( Figure 5 ) was also digested with NotI and Sac I endonucleases at 16°C overnight, and the ligation product was transformed into Escherichia coli DH5α, cultured upside down at 37°C overnight on LB medium containing 50 mg / L kanamycin, and the growth of resistant The plasmid was extracted from the colony and identified by enzyme digestion, and the plasmid that could obta...

Embodiment 3

[0049] Example 3. Construction of the expression vector pbin-NR-UΩ-NP1 of Chlorella ellipsoides nitrate reductase mutant

[0050] According to conventional techniques, the GUS gene on the pbin-NR-UΩGUS plasmid was partially digested with BamH I and SacI, and the vector was recovered. The target gene NP-1 (its sequence is as shown in SEQ ID NO: 12) has the restriction sites of BamH I and Sac I at both ends. The NP-1 gene of about 200 bp is recovered by enzyme digestion, and then ligated with the above-mentioned recovered vector overnight at 16°C , the ligation product was transformed into Escherichia coli DH5α, cultured upside down on LB medium containing 50mg / L ampicillin at 37°C overnight, and the resistant colony was identified by plasmid digestion, and a fragment of about 200bp (NP-1 gene) to construct the NP-1 plant expression vector pbin-NR-UΩ-NP1( Figure 9 ).

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Abstract

The invention constructs an expression vector suitable for expressing an exogenous gene in a chlorella nitrate reductase deletion mutant by using a Ubiquitin promoter and an omega enhancer as starting elements and the nitrate reductase gene NR. An objective gene is converted to the mutant by using an economic and simple chlorella PEG conversion method and employing the Chlorella ellipsoidea nitrate reductase deletion mutant as a receptor. The transgene chlorella wall breaking technology is optimized, the expressed exogenous gene has normal biological activity and is stably inherited in a culture medium without antibiotics. The results of the invention can high efficiently and safely be used to express the exogenous gene which does not suit to be expressed by the normal genetic engineering expression system (such as the escherichia coli and yeast) by using the genetic engineering technology and the Chlorella ellipsoidea nitrate reductase deletion mutant as a vector.

Description

technical field [0001] The invention relates to the field of plant genetic engineering. Specifically, it involves the establishment and application of a system for efficiently and safely expressing exogenous proteins that are not suitable for expression in conventional genetic engineering expression systems (such as Escherichia coli and yeast) using Chlorella ellipsoidal nitrate reductase deletion mutants. Background technique [0002] Chlorella (Chlorella) belongs to a class of single-celled eukaryotic microalgae belonging to the genus Chlorella of the Chlorella phylum. It has the characteristics of eukaryotic gene expression and is rich in protein, amino acids, vitamins, iron, calcium and other nutrients. It is considered It is an extremely valuable source of food and feed additives (Bricknell and Dalmo, 2005), and is easy to reproduce and cultivate, suitable for large-scale production. [0003] Based on the above-mentioned characteristics of Chlorella, using Chlorella as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N1/13C12N15/53A23L1/337A23L17/60
Inventor 胡赞民安洋赵世民孙勇如尹维波陈宇红胡军张丽华
Owner 北京中加保罗生物科技有限公司
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