摘要
以国审油茶(Camellia oleifera)良种‘华硕’种子为材料,在已构建的转录组和表达谱数据库基础之上,采用RACE技术,克隆获得油茶脂酰辅酶A脱氢酶基因的全长c DNA序列,命名为Co ACAD(基因登录号KJ910338)。该基因c DNA全长为2702 bp,含有2487 bp的开放读码框,编码828个氨基酸,分子量为92.4113 k D,理论等电点p I为8.47,具有2个比较明显的跨膜区和酪氨酸蛋白激酶活性位点LVHGDFRIDNLVF,存在5个亚结构域;在Co ACAD基因c DNA全长序列的基础上构建表达载体,其中原核表达载体在宿主细胞BL21(DE3)中成功诱导表达,获得表观分子量约为93 k D的目的蛋白;实时荧光定量PCR分析表明,Co ACAD基因在果实膨大期和成熟期上调表达,预示着Co ACAD基因可能在种子发育过程中参与能量供应过程的调控。
In this paper, using state trial oil-tea variety （ Camellia oleifera ＇ Huashuo＇ ） as material, a full- length cDNA of acyl-CoA dehydrogenase genes in seeds was cloned through RACE technique based on the transcriptome and expression profiling database of C. oleifera seeds. It was named as CoACAD （GenBank accession number K.1910338）. The cDNA of CoACAD gene was found to be 2702 bp with an open reading frame of 2487 bp encoding 828 amino acid residues. The molecular mass of the CoACAD protein was 92. 4113 kD with theoretical pI of 8.47. CoACAD protein had two obvious transmembrane domains and a typical tyrosine protein kinase （PTK） ac- tive site LVHGDFRIDNLVF, moreover, contained protein kinase domain, aminoglycoside phosphotransferase （APH） domain,ACAD_N domain, ACAD_C domain and ACAD Center domain. The expression vector of CoACAD were constructed successfully,and the BI21 （DE3）bacteria harboring pET30a-CoACAD was induced to express the recombinant protein which was about 93 kD. The relative expression abundance of CoACAD at 13 different develop- mental stages of C. oleifera seeds was analyzed using real-time quantitative PCR （qPCR）. The CoACAD gene were both up-regulated expression in the C. oleifera seeds enlargemental and mature period, which might be involved in regulation of energy supply at different developmental stages.
出处
《植物遗传资源学报》
CAS
CSCD
北大核心
2015年第5期1080-1088,共9页
Journal of Plant Genetic Resources
基金
国家自然科学基金(31170639
1070603)
湖南省自然科学基金(14JJ2104)
中南林业科技大学青年基金重点项目基金(QJ2011008A)
关键词
油茶
脂酰辅酶A脱氢酶
克隆
表达分析
Camellia oleifera
acyl-CoA dehydrogenase
clone
expression analysis
