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Chlamydomonas exogenous gene expression system based on blue light induction and application thereof

An exogenous gene and expression system technology, applied in the field of genetic engineering, can solve the problems of not developing a transgenic system, affecting the insertion and stability of the ble gene, and affecting the growth state of algae cells, etc., to achieve the effect of reducing production costs and simple operation of blue light induction

Active Publication Date: 2017-04-26
SHENZHEN UNIV
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Problems solved by technology

Experiments by Victoria Lumbreras et al. (1998) showed that the deletion RBCS2 After about 440bp of the upstream sequence of the promoter, it can be used ble 3-fold improvement in chemical efficiency, but not ble The expression efficiency of the gene may be that there are negative regulatory elements in the deleted sequence, which affects the ble Insertion and stabilization of genes in the genome [Lumbreras V., Stevens D. and Purton S.1998. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 14, 441-448】
RBCS2-HSP70 is currently a widely used promoter. It is a chimeric promoter of the small subunit promoter of 1,5-bisphosphate carboxylase and heat shock protein 70. It can be induced by strong light and heat shock. , can efficiently induce the expression of exogenous genes【Chaogang Wang, Zhangli Hu, Anping Lei, Baohui Jin . Biosynthesis of poly-3-hydroxybutyrate (PHB) intransgenic green algae Chlamydomonas reinhardtii. Journal of Phycology , 2010,46:396- 402.], but whether it is strong light irradiation or heat shock treatment will seriously affect the growth state of algae cells, and heat shock treatment is not suitable for large-scale cultivation of algae induction treatment
[0004] Blue light refers to light with a wavelength of 475-495nm. Using blue light receptors and interacting proteins in plants, a gene expression system based on blue light induction can be constructed. This system has been widely used in animal and plant cells [SilvanaKonermann, Mark D.Brigham, Alexandro E.Trevino et al. Nature, 2013,50(22):472-476], but no transgenic system based on blue light-induced regulation has been developed in algal cells

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  • Chlamydomonas exogenous gene expression system based on blue light induction and application thereof
  • Chlamydomonas exogenous gene expression system based on blue light induction and application thereof
  • Chlamydomonas exogenous gene expression system based on blue light induction and application thereof

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Embodiment 1

[0016] Example 1 The expression system of Chlamydomonas exogenous genes induced by blue light and the two vector skeletons required for the construction of the Chlamydomonas exogenous gene expression system based on blue light induction are based on the vector pH124 (refer to ZL201110070784.1) preserved in our laboratory Transformed from above. The transformation process of the carrier is as follows.

[0017] First, the heat-shock-activated vector pH124 was transformed into a constitutive expression vector pDb124. Chlamydomonas PSaD It has the characteristics of high constitutive expression, so the 5' end and 3' end sequences of the gene are selected to mediate the constitutive expression of the light-regulating protein. by PCR at 5' PSaD introduced in the sequence not I and Pma CⅠ restriction site, through not I and Pma Restriction digestion and ligation of the CI site made the pH124 promoter of the vector HSP70-RBCS2 Sequence replaced with 5' of 822bp PSaD s...

Embodiment 2

[0025] Example 2 Transformation of Chlamydomonas luciferase gene expression vector and screening of transgenic algae Chlamydomonas reinhardtii CC-849 was cultured to the logarithmic phase in TAP culture medium, and the number of cells was about 1×10 6 cells / mL, collected by centrifugation and resuspended with TAP medium, adjust the cell concentration to 1×10 8 cells / mL; pipette 300 μL of the suspension into two 5mL test tubes (containing sterilized quartz sand), cut the pDb124-BD-CIB1 expression vector enzyme into lines, take 1 μg-2 μg into the test tube, After the CC-849 / quartz sand / carrier DNA mixture was shaken rapidly for 15 seconds; transfer the mixture to a 25 mL centrifuge tube with a screw cap, add 10 mL of sterilized TAP culture solution, and culture overnight on a shaking shaker at 40 rpm at room temperature (light condition is 90μE / m 2 / s); collect the cells by centrifugation at room temperature, remove the supernatant, resuspend with 0.5 mL TAP, add 3.5 mL 0.5%...

Embodiment 3

[0027] Example 3 Blue-light-induced expression of exogenous reporter gene in Chlamydomonas reinhardtii. Transgenic algae and CC-849 were inoculated in equal amounts in culture flasks to ensure the same algae biomass; then the transgenic algae and CC-849 were cultured under continuous red light to In the logarithmic growth phase, the expression level of the reporter gene was detected as a reference; after that, it was cultured under continuous blue light irradiation, and the algal cells of the two genotypes were collected after 24 hours to detect the expression level of the exogenous reporter sequence, and it was found that the transgene Algal strains have transcription of exogenous reporter sequences, and are significantly induced by blue light, while blue light induction has no effect on CC-849. It shows that the Chlamydomonas exogenous gene expression system based on blue light induction has been successfully constructed, which can be used for the high-efficiency expression o...

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Abstract

The invention discloses a chlamydomonas exogenous gene expression system based on blue light induction and an application thereof. Expression system elements include a light receptor cryptochrome CRY2 gene and an interacting protein CIB1 gene, a GAL4 DNA binding structural domain BD and an activation domain VP64 having transcriptional activation of an active herpes simplex virus, a nuclear localization sequence NLS, a UAS sequence which can be combined with the GAL4 transcription factor DNA binding structural domain BD, and an exogenous gene, wherein CRY2-BD, CIB1-VP64-NLS and a UAS-exogenous gene are connected together and are integrated into a nuclear genome of chlamydomonas through genetic transformation. Single color light-blue light is used as an inducing factor for inducing the expression of the exogenous gene, and the damage of algae cells caused by heat shock induction is overcome, and the influence of various chemical inducers on the metabolism of the algae cells is overcome.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to an exogenous gene expression system induced by blue light and its application in Chlamydomonas gene expression regulation and transgenic Chlamydomonas bioreactor. Background technique [0002] Transgenic technology was first developed in Escherichia coli in the 1970s. With the rapid development of molecular biology, the high-efficiency expression of foreign genes has become an important production method for genetic engineering drugs and industrial and agricultural raw materials. Algae are very suitable for the construction of transgenic algae bioreactors because they have the characteristics of rapid microbial growth and can synthesize target products through photosynthesis. [0003] Chlamydomonas reinhardtii is one of the few organisms with three sets of genomes capable of genetic transformation【Lumbreras, V., Purton, S., 1998. Recent advances in Chlamydomonas transgenics. Pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79
CPCC12N15/79
Inventor 王玉婷蒋欣芩李辉胡章立
Owner SHENZHEN UNIV
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