Coexpression In Pichia Pastoris Of ’Newhall’ Navel Orange CsKO Or CsKAO With CsCPR And Research Of The Expressed Products

Ent-kaurene oxidase KO and ent-kaurenoic acid oxidase KAO are important limiting enzymes in the process of gibberellin biosynthesis.In recent years, more and more genes coding these two P450s have been cloned and functional verification, such as Arabidopsis, Rice, Physcomitrella patens, but there is little research in citrus.In order to study and validate the citrus enzyme function,we separate the Navel Orange ent-kaurene oxidase CsKO, ent-kaurenoic acid oxidase CsKAO and its own NADPH-cytochrome C reductase CsCPR. By using Pichia pastoris X-33coexpression’s way to prove that the two kinds gene of encoding enzyme function. The main research results:1.Isolation and cloning of ’Newhall’Navel Orange NADPH cytochrome reductase encoding gene CsCPR using mature leaves of ’Newhall’ navel orange.The full-length of CDS is2148bp, encoding715amino acids, predicted molecular weight and isoelectric point are79.2kDa and5.32respectively.And using cocoa and poplar CPR gene sequences blast CsCPR gene sequences find that their homologous rate can reach83%and82%respectively. CsCPR can catalyze the reaction of oxidized cytochrome C under the conditions of NADPH’s existence, then its A550value changes.2.Using the electrical transformation and LiCl chemical transformation can obtain double transformants with a same vector of two different genes or different resistance and different gene vectors.And transform method and positive clone selection method are selected and optimized. We found that the double transformants with same resistance vector can be obtained by electrical transformation method, and the double transformants with different resistance vector can be obtained by LiCl chemical transformation.3.The research optimize the expression condition of the Pichia pastoris expression of P450oxidase protein.We text the total protein content and P450protein content of microsomal protein expression in different culture, the research find that4days,pH=5,0.75%methanol concentration can obtain the highest protein content. Other expression conditions were also studied by orthogonal experimental analysis, found the impact on protein expression of these factors: temperature﹥content of YNB﹥BMGY:BMMY﹥methanol concentration.4.With different substrates in the two enzyme reaction system, GC--MS analysis of the reaction product verificate their functions.Under co-expression conditions, Double transformants in Pichia pastoris CsKO can catalyze the oxidation reaction from ent-kaurene or ent-kaurenol to ent-kaurenoic acid. Here the ent-kaurene can be the standard,can also be the ent-kaurene in Cryptomeria japonica. CsKAO in double transformants can also catalyze the oxidation reaction of ent-kaurenoic acid or GA12-aldehyde to GA12.