黑茶藨子(Ribes nigrum L.)含Cytb_5结构域脱氢酶基因的克隆与功能研究
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摘要
含Cytb_5结构域的脱氢酶是近些年发现的一类新的小家族蛋白,这些蛋白都含有一个标志Cytb_5蛋白特征的血色素结合区:“His-Pro-Gly-Gly domain”;同时,它们还含有膜结合脱氢酶所必需的三个保守的“His Box”,通常为HisⅠ区HK_(3-4)H、HisⅡ区HX_(2-3)HH和HisⅢ区(H/Q)X_(2/3)HH。现已知:Δ6-、Δ5-和Δ4-脂肪酸脱氢酶、脂肪酸羟基化酶以及Δ8-鞘脂脱氢酶是该家族蛋白的主要成员。在植物中,此类蛋白研究最多的是△6-脂肪酸脱氢酶和Δ8-鞘脂脱氢酶,前者是γ-亚麻酸生产中的关键酶,后者可能涉及植物的细胞程序性死亡和抗逆作用,因此具有重要研究意义。本研究以富含γ-亚麻酸的植物黑茶藨子(Ribes nigrum L.)为材料,利用基因组步移方法从黑茶藨子DNA中克隆获得了多个含Cytb_5结构域的脱氢酶基因,通过酿酒酵母异源表达初步鉴定了基因功能,并进行了基因间功能差异和进化关系等方面的分析研究。主要研究结果如下:
1 含Cytb_5结构域脱氢酶基因的克隆
根据已知的Δ6-脂肪酸脱氢酶和Δ8-鞘脂脱氢酶保守区序列设计简并引物,以黑茶藨子DNA为模板,通过PCR扩增技术,共获得三个约1032bp大小的脱氢酶相关基因的中间片断。在此基础上,利用基因组步移技术,获得两个含Cytb_5结构域的脱氢酶相关基因的全长序列RnCyDA、RnCyDB,以及一个含有ATG起始密码子的含Cytb_5结构域的脱氢酶相关基因片断RnCyDC。
RnCyDA、RnCyDB全长1347bp,编码448个氨基酸,RnCyDB第54位含一个终止子;RnCyDC片断长1140bp。RnCyDA、RnCyDB和RnCyDC具有共同特点:在第41aa位点处具有Cytb_5蛋白标志的HPGG domain,在159～163aa、196～200aa、375～
Cytochrome b_5 fusion desaturases represent a new class of family proteins which share the same characteristics fusing a Cytochrome b_5 domain HPGG involved in heme-binding and owning a conserved tripartite motif of membrane-bound desaturases, HX_(3-4)H, HX_(2-3)HH and (H/Q)X_(2/3)HH. 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 b_5 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 b_5 fusion desaturase genesThe DNA fragments of putative Cytochrome b_5 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 Cytb_5 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
1 含Cytb_5结构域脱氢酶基因的克隆
根据已知的Δ6-脂肪酸脱氢酶和Δ8-鞘脂脱氢酶保守区序列设计简并引物,以黑茶藨子DNA为模板,通过PCR扩增技术,共获得三个约1032bp大小的脱氢酶相关基因的中间片断。在此基础上,利用基因组步移技术,获得两个含Cytb_5结构域的脱氢酶相关基因的全长序列RnCyDA、RnCyDB,以及一个含有ATG起始密码子的含Cytb_5结构域的脱氢酶相关基因片断RnCyDC。
RnCyDA、RnCyDB全长1347bp,编码448个氨基酸,RnCyDB第54位含一个终止子;RnCyDC片断长1140bp。RnCyDA、RnCyDB和RnCyDC具有共同特点:在第41aa位点处具有Cytb_5蛋白标志的HPGG domain,在159～163aa、196～200aa、375～
Cytochrome b_5 fusion desaturases represent a new class of family proteins which share the same characteristics fusing a Cytochrome b_5 domain HPGG involved in heme-binding and owning a conserved tripartite motif of membrane-bound desaturases, HX_(3-4)H, HX_(2-3)HH and (H/Q)X_(2/3)HH. 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 b_5 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 b_5 fusion desaturase genesThe DNA fragments of putative Cytochrome b_5 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 Cytb_5 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
引文
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