以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据GenBank中已经发表fad2基因的同源序列,利用PCR技术克隆得到基因片段。在GenBank中Blast(GenBank登录号AY957393)所得基因片段和同属豆科的Glycine max Gmfad2-2a同源性高...以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据GenBank中已经发表fad2基因的同源序列,利用PCR技术克隆得到基因片段。在GenBank中Blast(GenBank登录号AY957393)所得基因片段和同属豆科的Glycine max Gmfad2-2a同源性高达88%,位于fad2基因编码区中部。将所得片段经BamHI和SacI酶切后插入表达载体质粒pBI121,构建了反义表达载体pBI121fad2,并利用农杆菌介导法转入烟草叶片,获得了抗卡那霉素和氨苄青霉素的再生烟草植株。初步分析结果表明:与对照烟草相比,转基因烟草种子脂肪酸含量没有明显差异,而亚油酸则减少10.3%。展开更多
The fatty acid desaturase 2(fad2) gene was proven to be a major locus for high oleic acid(C18:1).Brassica napus is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa and Brassica ole...The fatty acid desaturase 2(fad2) gene was proven to be a major locus for high oleic acid(C18:1).Brassica napus is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa and Brassica oleracea.B.napus contains multiple copies in genome for most of the genes,including fad2 genes.The research cloned nine fad2 genes from 3 varieties of B.rapa and 3 varieties of B.oleracea,respectively.Alignment of the nine fad2 sequences from B.rapa and B.oleracea detected 6 single nucleotide polymorphic sites,which resulted in 6 amino-acid substitutions.The nucleotide substitutions at position 743 bp in the fad2-A gene and position 947 bp in the fad2-C gene were used as 3' end of allele-specific primers.In use of the allele-specific primers to amplify fad2 gene,we could identify if the fad2 gene originated from A genome or C genome.Besides,the research found that fad2 genes in C genome are more conserved in evolutionary process than those in A genome.The fad2 expression data reported in this study revealed that fad2-A from B.rapa was not only expressed in siliques same as fad2-C from B.oleracea,but also expressed in a high level in stems.Not even the less,fad2 gene from B.napus was expressed higher in roots and flowers.All these results provided evidences that fad2,though it was expressed differently in B.rapa and B.oleracea,but it was regulated by the same approach in B.napus.展开更多
文摘以我国重要的生物能源灌木——中间锦鸡儿枝叶和种子为材料,根据GenBank中已经发表fad2基因的同源序列,利用PCR技术克隆得到基因片段。在GenBank中Blast(GenBank登录号AY957393)所得基因片段和同属豆科的Glycine max Gmfad2-2a同源性高达88%,位于fad2基因编码区中部。将所得片段经BamHI和SacI酶切后插入表达载体质粒pBI121,构建了反义表达载体pBI121fad2,并利用农杆菌介导法转入烟草叶片,获得了抗卡那霉素和氨苄青霉素的再生烟草植株。初步分析结果表明:与对照烟草相比,转基因烟草种子脂肪酸含量没有明显差异,而亚油酸则减少10.3%。
基金Supported by the National Natural Science Foundation of China (31301357)the Natural Science Foundation of Jiangsu Province,China (BK20130719)
文摘The fatty acid desaturase 2(fad2) gene was proven to be a major locus for high oleic acid(C18:1).Brassica napus is an amphidiploid species originating from a spontaneous hybridization of Brassica rapa and Brassica oleracea.B.napus contains multiple copies in genome for most of the genes,including fad2 genes.The research cloned nine fad2 genes from 3 varieties of B.rapa and 3 varieties of B.oleracea,respectively.Alignment of the nine fad2 sequences from B.rapa and B.oleracea detected 6 single nucleotide polymorphic sites,which resulted in 6 amino-acid substitutions.The nucleotide substitutions at position 743 bp in the fad2-A gene and position 947 bp in the fad2-C gene were used as 3' end of allele-specific primers.In use of the allele-specific primers to amplify fad2 gene,we could identify if the fad2 gene originated from A genome or C genome.Besides,the research found that fad2 genes in C genome are more conserved in evolutionary process than those in A genome.The fad2 expression data reported in this study revealed that fad2-A from B.rapa was not only expressed in siliques same as fad2-C from B.oleracea,but also expressed in a high level in stems.Not even the less,fad2 gene from B.napus was expressed higher in roots and flowers.All these results provided evidences that fad2,though it was expressed differently in B.rapa and B.oleracea,but it was regulated by the same approach in B.napus.
