Cloning Laccase Gene From Edible Fungi And Established The System Of Transformation Lactic Acid Bacteria By Electroporation

Silage is an important material base for the development of animal husbandry, lactic acid bacteria determines the quality of Silage, with the rapid development of livestock industry in China, the demand of the silage is increasing, therefore, improve the utilization rate of feed is the key to solve the shortage of fodder. Laccase has a strong ability to degrade lignin, so, the degradation of cellulose can be fully. In recent years, the function of laccase degradation has attracted more and more attention, therefore, Lactobacillus buchneri as experiment materials, by using biophysical techniques to introduce genes intends to build a new lactic acid bacteria. In order to the metabolic product of bacteria have both lactate and laccase, not only improving the quality of the feed, but also increase the efficiency of feed.The main conclusions are as follows:1, Select a variety of edible fungi as tested material, according to RB light blue color intensity to choose three kinds of edible fungi, using ABTS method mesure the activity of laccase, enzyme activity of Pleurotus eryngii is324.07U/L, Pleurotus ostreatus is43.98U/L, Pleurotus nebrodensis is17.36U/L.2, Design primers according to the conserved sequence of pleurotaceae laccase gene, We obtained three kinds edible fungi laccase gene by using PCR, The length of amplification fragment was2606bp (GeneBank registration number were KC789845, KC789846, KC789847), through the intron and exon analysis of gene sequence, we achieved coding cDNA of laccase, It contains one open reading frame (ORF), which encodes for a polypeptide containing531amino acids. To predict three kinds of edible fungi laccase protein molecular weight were56682.0,56692.0,56727.1, Acid isoelectric point, pI was4.56, all contains nine possible N-glycosylation sites.3, Obtained the cDNA sequence of Lacc1from Pleurotus eryngii by RT-PCR method. By plasmid extraction, enzyme digestion, purification, connecting with vector plasmid pMG36e, obtained the recombinant plasmid pMG36e-Laccl.4, Explored optimal condition affect lactobacilli electricity transformation, the recombinant plasmid was transformed into Lactobacillus buchneri, when the electric field intensity is1.75kV,1.5h to medium SMRS recovery, obtained the high efficiency of conversion by electroporation transformation.