Isolation And Characterization Of Cytochrome B₅ Fusion Desaturase Gene From Black Currant (Ribes Nigrum L.)

Cytochrome b₅ fusion desaturases represent a new class of family proteins which share the same characteristics fusing a Cytochrome b₅ domain HPGG involved in heme-binding and owning a conserved tripartite motif of membrane-bound desaturases, HX_3-4H, HX_2-3HH and (H/Q)X_2/3HH. To date, Δ6-, Δ5- and Δ4-fatty acid desaturases, hydroxylases and Δ8-sphingplipid desaturase have been found in the family. A6-fatty acid desaturase and A8-sphingplipid desaturase in plant had been studied because the former is the key desaturase in producing of GLA and the latter is likely to involve in the programmed cell death (PCD) and the resistance of plant. In our studies, several Cytochrome b₅ fusion desaturase genes were cloned from genomic DNA of black currant (Ribes nigrum L.) by the genomic walking method and expressed in yeast (Saccharomyces cerevisiae) to determine their functions. Some reasons about different functions and possible evolutionary relationships among these genes were discussed. The main results are provided as following:1 Cloning of Cytochrome b₅ fusion desaturase genesThe DNA fragments of putative Cytochrome b₅ fusion desaturase genes were amplified from the genomic DNA of black currant leaves by using degenerated primers designed according to the sequence of conserved histidine boxes of known Cytb₅ fusion desaturases. Three amplified products (about 1032 bp) were cloned and 5' and 3' extension of these fragments were obtained by the genomic walking method. Two full-length sequences, 1347 bp in length, named RnCyDA and RnCyDB, and a fragment with 1140 bp, named RnCyDC with ATG start codon thus were obtained.RnCyDA encodes 448 aa. RnCyDB has a stop codon at 54th aa. The deduced amino acid sequences of RnCyDA, RnCyDB and RnCyDC contain cytochrome b5-like heme-binding domains at their N-terminus and the diagnostic three "histidine box" of membrane-bound desaturases.2 Site-directed mutagenesis of RnCyDB and construction of chimeric genes RnCyDCA, RnCyDCB.The site-directed mutagenesis was performed for RnCyDB by using overlap extend method and " 160TAA " which is encoded for stop codon was mutated to be " 160CAA " encoding amino acid "Q" . The mutated RnCyDB was named RnCyDB 1. The 208bp terminal sequence of RnCyDA and RnCyDB were linked with RnCyDC, respectivly and two chimeric genes RnCyDC A and RnCyDCB with entire 1347bp ORF were obtained.3 Bio-information analysisPrimary bioinformation of RnCyDA, RnCyDB 1 and RnCyDCA/B were obtained by using some softwares in some web sites. Based on the obtained information, membrane topology models were proposed for RnCyDA, RnCyDB 1 and RnCyDCA/B.4 Function characterization of cloned Cytochrome b$ fusion desaturase genes The open reading frames (ORFs) of RnCyDA, RnCyDB 1 and RnCyDCA/B werecloned into the yeast expression vector pYES2 (Invitrogen) by using the Hindlll and Xbal restriction sites. The constructed vectors were used to transform yeast (Saccharomyces cerevisiae) strain INVSc I by using the lithium acetate method and the recombinant yeast cells were selected on a uracil-deficient medium. The expression of the transformed gene controlled by the GAL promoter was induced by adding galactose (2%, w/v) in suspension culture. Sphingolipid LCB and/or total fatty acids of cultured yeast were extracted and their methyl esters were analyzed by GC-MS.The presence of additional desaturated 8-LCBs is observed in yeast cells expressing RnCyDA, but not in the control with the empty vector. It might be predicted that the black currant RnCyDA encodes a A8-sphingplipid desaturase. GC-MS analysis of FAME (fatty acid methyl esters) of cultured yeast expressing RnCyDCA/B revealed that novel fatty acid peaks corresponding to GLA and OTA were detected, but absent in...