Dual-luciferase monitoring plasmid for detecting living yeast cell UPR level, construction and application thereof
A dual-luciferase and yeast cell technology, which is applied in the field of dual-luciferase monitoring plasmids and its construction and application, can solve the unavoidable interference of marker protein folding, the inability to detect high-throughput living cells, and the low accuracy of plasma determination, etc. problem, to achieve the effect of shortening the detection cycle, short detection cycle and cumbersome operation
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Embodiment 1
[0055] Construction of UPR detection plasmid pUST-08
[0056] Using conventional gene cloning techniques, the operable promoter Gal10p, luciferase reporter gene Rluc, HAC1 promoter 5'UTR sequence Hac1p, HAC1 intron Hac1i and its 3'UTR sequence, and luciferase reporter gene Fluc were cloned into yeast in sequence. In the low copy shuttle plasmid YCplac33 (such as figure 2 shown).
[0057] 1. Design primers
[0058] Gal10p-F: ACGCCAAGCTTGATCAAAAATCATCGCTTCG;
[0059] Gal10p-R: GCTCTAGACTCGACTCAAAAATTCTTAC;
[0060] Rluc‐Hac1p‐F: GCTCTAGAGTCCACCAATGACTTCGAAA;
[0061] Rluc‐Hac1p‐R: CGGGATCCTAGGTGGGGGAGGAGGAGGTTCGACATTTGTTCATTTTTGAG;
[0062] Hac1i‐F: GGGGTACCTATAGCGGGAAACAGTCTAC;
[0063] Hac1i‐R: CCACCAACAGCGATAATAAC;
[0064] Fluc‐F: TCAAAAATGAACAAATGTCG;
[0065] Fluc‐R: GGGGTACCATGACTCGAGCACGTGTTAC.
[0066] 2. Using plasmid YEP51-LIPIN1 as template, Gal10p‐F and Gal10p‐R as amplification primers (Gal10p‐F: ACGCCAAGCTTGATCAAAAATCATCGCTTCG; Gal10p‐R: GCTCTAGACTCGACTC...
Embodiment 2
[0080] Detecting Alternative Splicing of HAC1mRNA Using UPR Routine Detection Technology RT-PCR
[0081] Using conventional RT-PCR technology to detect the splicing rate of HAC1mRNA in wild-type yeast treated with 0.5 μg / mL tunicamycin, 2 μg / mL tunicamycin, 0.1 mM DTT, and 0.5 mM DTT
[0082] 1. Preferably use primers
[0083] ACT1-F:GCTTTGTTCCATCCTTCTG, as shown in SEQ ID NO:9;
[0084] ACT1-R:GAAACACTTGTGGTGAACG, as shown in SEQ ID NO:10;
[0085] HAC1-F: AGGAAAAGGAACAGCGAAGG, as shown in SEQ ID NO: 11;
[0086] HAC1-R:GAATTCAAACCTGACTGCGC, as shown in SEQ ID NO:12.
[0087] 2. According to the conventional RT-PCR technique to detect the splicing rate of HAC1, the steps are as follows: (1) clone the wild-type yeast BY4741a, pick it into 3mL SC-URA liquid medium as the seed fungus, and culture it overnight at 30°C and 180rpm; (2) ) the next day, the above seed bacteria were respectively transferred to fresh 6mL SC-URA liquid medium to ensure that the initial bacterial con...
Embodiment 3
[0090] Confirmation that hac1Δ and ire1Δ are ultra-sensitive UPR strains
[0091] IRE1-mediated activation of HAC1 mRNA splicing is one of the important pathways of UPR. The synthesized HAC1 can improve the protein folding ability of endoplasmic reticulum by activating the expression of downstream UPR-related genes to relieve endoplasmic reticulum stress. The deficiency of HAC1 or IRE1 will lead to the failure of the endoplasmic reticulum stress rescue mechanism of IRE1 / HAC1 in response to environmental stress, and aggravate the accumulation of unfolded proteins in the endoplasmic reticulum. To confirm this argument, HAC1 and IRE1 deletion mutants were tested for sensitivity to UPR-inducing drugs.
[0092] 1. Pick the strains to be tested (BY4741a, hac1Δ, ire1Δ) into 3mL SC liquid medium as seed bacteria, and culture overnight at 30°C and 180rpm.
[0093] 2. Take about 1×10 7 of yeast cells (OD 600 ≈1), after 10-fold serial dilution (dilution of 5 gradients), take 5ul of th...
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