The Expression Of Two Linoleate Isomerase Genes In Pichia Pastoris

Conjugated Linoleic Acid (CLA) have attracted great interest because of their potential health properties. They have been reported to have the potential of anticarcinogenic, antiatherosclerotic, antidiabetogenic, anti-oxidative, anti-bacterial, cholesterol depressing, as well as the ability of growth promoting in animal and human. They are rarely found in whole food. Thus, it is of great value to construct genetic engineering bacteria in order to get safe and highly efficient CLA.In this thesis, alcohol oxidase gene of Pichia pastoris GS115was replaced by the linoleate isomerase gene from Lactobacillus acidophilus AS1.1854or Lactobacillus plantarum AS1.555, to form recombined Pichia pastoris, the expression of which was induced by methyl alcohol.Linoleate isomerase genes were analysed by BioEidt to predict restriction sites. Multiple clone sites of plasmid pPICZaA were consulted. One pair of Lactobacillus acidophilus primers and another pair of Lactobacillus plantarum primers were designed and synthesized separately.Plasmid pMD19T-PDI, which contained the linoleate isomerase gene from Lactobacillus acidophilus AS1.1854, was used as template. Target gene was amplified by polymerase chain reaction (PCR) with the above-mentioned primers, and a fragment of about1760bp was obtained. Target gene and pMD19T vector were ligated, and then transformed into E. coli DH5a. The recons were screened by Ampicillin and confirmed by the methods of restriction enzymes and PCR in the following step. The positive plasmid was named pMD19T-LAI. Restriction enzymes Xho I and Xba I were used to digest positive plasmid pMD19T-LAI and shuttle plasmid pPICZaA separately. Different target gene fragments were separated by gel electrophoresis and retrieved from gel. Then they were ligated and transformed into E. coli DH5a. Recons were screened by zeocin and confirmed by restriction enzyme and by PCR mehtods. Recombinant plasmids were sequenced. Proper sequence plasmid was named pPICZaA-LAI. Plasmid pPICZaA-LAI was linearized, and the linear pPICZaA-LAI was transformed into Pichia pastoris GS115by chemical method or by electrotransformation. Recombinant Pichia pastoris were screened by zeocin. Genome of recombinant Pichia pastoris was extracted and confirmed by PCR method. Positive recombinant Pichia pastoris GS115/LAI was fermented in Shake flask. Yeast was separated from suspension. Supernatant was detected by SDS-PAGE, and enzyme activity of the supernatant was0.107U. Related reports of Lactobacillus acidophilus linoleate isomerase activity in recombinant Pichia pastoris have not been seen up to date.Lactobacillus plantarum genome was used as template to amplify the linoleate isomerase gene by PCR, with the above-mentioned primers. A fragment about1710bp was obtained. Target gene and pMD19T vector were ligated and transformed into E. coli DH5a. Recons were screened by Ampicillin and confirmed by restriction enzyme method and by PCR method. Recombinant plasmids were sequenced, and the proper sequence was named pMD19T-LPI. Restriction enzymes EcoR Ⅰ and Xba Ⅰ were used to digest positive plasmid pMD19T-LPI and shuttle plasmid pPICZaA separately. Different target gene fragments were separated by gel electrophoresis and retrieved from gel. Then they were ligated and transformed into E. coli DH5a. Recons were screened by zeocin and confirmed by restriction enzyme and by PCR mehtod. Recombinant plasmids were sequenced. Proper sequence plasmid was named pPICZaA-LPI. The pPICZaA-LPI was linearized, and the linear pPICZaA-LPI was transformed into Pichia pastoris GS115by chemical method or by electrotransformation. Recombinant Pichia pastoris were screened by zeocin and confirmed by PCR method. Positive recombinant Pichia pastoris GS115/LPI was fermented in Shake flask. Yeast was separated from suspension. Supernatant was detected by SDS-PAGE. Recombinase activity of supernatant of recombinant Pichia pastoris was0.130U.