[Re-cloning of THP gene and construction of high efficient expression yector of Volvariella volvacea].
PCR technique was used for amplifying THP gene in an unknown vector with primer AFP1 and AFP2. Then THP gene was ligated to pGEM T-Vector to be the plasmid pGTHP4. The plasmid pCAMBIA1301 was digested with restriction enzyme BstE II and Nco I, and digestion product was separated with 1% of agarose gel, then big fragment containing promoter was isolated and purified with the Agarose Gel DNA Extraction Kit. At the same way, the plasmid pGTHP4 was digested with restriction enzyme BstZ I and Nco I, and the small fragment containing THP gene was purified from 1% agarose gel with the Agarose Gel DNA Extraction Kit. The big fragment and the small fragment were ligated at Nco I digested cohesive-end. The ligation product was re-ligated to be cyclic plasmid by addition to a specific adapter, resulting in the pCTH823, a expression vectorof V. volvacea.
Guo
Liqiong
L
National Key Biotechnology Laboratory for Tropical Crops, China Academy of Tropical Agricultural Sciences, Haikou 571101, China.
Lin
Junfang
J
Chen
Shoucai
S
Xiong
Sheng
S
D000363:Agaricales / Q000235:genetics*; D021301:Antifreeze Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data
D021301:Antifreeze Proteins; D004247:DNA