Bay scallop heat shock protein 70 gene promoter and application thereof
A heat shock protein and promoter technology, applied in the fields of molecular biology and genetic engineering, can solve the problem of no heat-inducible promoter, etc., and achieve the effect of strong promoter activity
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Embodiment 1
[0030] Example 1 RT-PCR detection of heat-stressed scallop heat shock protein 70mRNA expression
[0031] Bay scallops for experiments were purchased from Qingdao Aquatic Products Market, and kept in seawater at (17±1)°C for a week, and then subjected to continuous high temperature stimulation experiments. 5 scallops were taken as the blank group, while the other 25 scallops were placed in seawater at 28°C for heat shock as the treatment group. At 1h, 2h, 4h, 8h and 12h after stimulation, the gills of scallops were frozen and stored at -80°C for RNA extraction. Using primer oligo dT (18) and reverse transcriptase M-MLV at 42°C for 1h. With the scallop housekeeping gene β-actin as an internal reference, the mRNA expression of the AiHSP70 gene was detected by RT-PCR (see figure 1 ).
[0032] The result is as figure 1 As shown, after heat shock at 28°C for 1 hour, the expression level of heat shock protein 70 mRNA in bay scallops rose rapidly and reached the maximum value, wh...
Embodiment 2
[0033] Example 2 Cloning of the promoter of the heat shock protein 70 gene of bay scallop
[0034] 1. Using the restriction enzymes (Dra I, EcoR V, PvμII & StμI) of the GenomeWalkerUniversal Kit (Catalog No. 638904) of Clontech Company (www.Clontech.com) to construct four DNA endonuclease libraries.
[0035] 2. Use two rounds of two-step PCR method to clone the 5'-end sequence, the specific steps are as follows:
[0036](1) The total system for the first round of PCR is 50 μl, including 38.5 μl sterile double distilled water, 5 μl 10×PCR buffer, 3 μl dNTP (10 mM), 1 μl linker primer (AP1: 5'-GTAATACGACTCACTATAGGGC-3', 10 μM), 1 μl gene Specific primer (SEQ ID NO.2, 10 μM)), 0.5 μl rTaq polymerase (TaKaRa) and 1 μl restriction library DNA constructed as described in Step 1 were used as templates. The PCR program is: 7 cycles: 94°C for 25s, 72°C for 3min; 32 cycles: 94°C for 25s, 67°C for 3min; 1 cycle: 67°C for 7min.
[0037] (2) The second round of two-step PCR uses the PCR ...
Embodiment 3
[0085] Example 3 Bay Scallop Heat Shock Protein 70 Gene Promoter (pAiHSP70) Activity Detection
[0086] 1. Construction of pGL-3-Basic-pAiHSP70 recombinant vector: 1) Synthesis of primers SEQ ID NO.4 (5'-GGTACCATAACGGCTTGCCATACTAAC-3') and SEQ ID NO.4 (5'-GGTACCATAACGGCTTGCCATACTAAC-3') and SEQ ID NO. ID NO.5 (5'-AGATCTTTGCTAAAAACAAAAACGAAAT-3'), was amplified by PCR with Takara high-fidelity Taq enzyme. The reaction system with a total volume of 50 μl contains 38.5 μl sterile double distilled water, 5 μl 10×PCR buffer, 3 μl dNTP (10 mM), 1 μl primer SEQ ID NO.4 (10 μM), 1 μl SEQ ID NO.5 (10 μM) ), 0.5 μl high-fidelity Taq polymerase (TaKaRa) and 1 μl whole genome DNA as template (50ng / ul). The PCR program is: 1 cycle: 94°C for 5 min; 32 cycles: 94°C for 30 s, 55°C for 30 s, 72°C for 90 s; 1 cycle: 72°C for 10 min. PCR products were purified using a gel extraction kit (TaKaRa).
[0087]2) The recovered PCR product and the empty promoter-free vector pGL-3-Basic (Promega) wer...
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