The Preliminary Study On The Gene Cloning Of Pectin Esterase Of The Lycopersicon Esculentum And Its Gene Expression In The Pichia Pastoris

Low methoxyl pectin is more widely used in the food, medical and other industry than the high methoxyl pectin. Being a kind of pectin complex Enzymes, and as a high-efficiency biocatalyst, pectin esterase (Pectin Methyl esterase, Pectin demethoxylase or Pectin Methoxylase, E.C.3.1.1.11), with different origins, can attack the methoxy from the reduced end of pectin or from the adjacent carboxyl, a demethylation reaction that transform the high methoxyl pectin to the low methoxyl pectin. Pectin esterase can be extracted from some leaf mold and bacteria, as well as from a wide variety of plants. Its utilization in the production of low methoxyl pectin saves some disadvantages such as heavy pollution, high costs and poor circularity seen in the methods of acidization, alkalifying process and amidation. Therefore, using enzyme-method to produce the pectin has become a hot spot in the research of Low methoxyl pectin.So far, there have been many reports on the research of pectin esterase, some of which have been successfully used in the cloning of the pectin esterase from Satsuma orange, tomato, bean sprout and other biological organism. But none of them was given further analysis. The present research chooses the fruits, young leaf and bud in different maturation stage of Lycopersicon esculentum grown in South America for the extraction of the total RNA, and then uses specific primers for RT-PCR reaction, thus amplifying the full length cDNA of pectin esterase gene, which is linked to the plasmid vector for sequence analysis. The research also constructs the recombinant expression plasmids pPICZaA, and transforms the Intact Yeast Cells for the Expression of Exogenous Genes to testify the activity of pectin esterase excreted from the transformed yeast Pichia pastoris. The research mainly includes:1. Extraction and Identification of the total RNA of the Fruits, young leaves and buds of Lycopersicon esculentum.2 . Reverse transcription of mRNA from the pectin esterase of Lycopersicon esculentum into the first-strand cDNA and the Design of Specific Primer for PCR Amplification, thus obtaining the gene fragment of the tomato pectin esterase.3. Construction of vector PT-PEM, extraction of plasmid,. and double Restriction enzyme, PCR reaction and sequence analysis to testify the success of the gene extraction from the pectin esterase of Lycopersicon esculentum .4. Construction of recombinant plasmid pPICZαA-PME; Transformation of Pichia pastoris GS-115; screening the Recombinant Yeast Cell from the selective YEPD solid culture medium containing the Zeocin?, and extracting the DNA in recombinant plasmid for PCR reaction to determine the gene fragment of pectin esterase contained in the recombinant plasmid gene.5. Expression induced by carbinol for the alien gene of pichia pastoris; testing the activity of pectin esterase in culture supernatant.Through the experiments, we get a pectin esterase gene with 1942bp long. After endonuclease digestion, cataphoresis, sequence analysis, and BLAST homology retrieval in Nr (http://www.ncbi.nim.nih.gov/blast) of NCBI (National Center for Biotechnology Information), it is of the same with the reference sequence. By linking the gene segment with the plasmid pPICZαA, we get recombinant plasmid pPICZαA-PME. After transforming the recombinant yeast, and induceing it, and testing the pectin esterase activity of supernatant ferment, we find that there is no activity in our expression of solanum pectin esterase.