从东方山羊豆(Galega.orientalisL.)中克隆出的GoMIPS(肌醇-1-磷酸合成酶)基因序列设计合成1对带有NcoI和SpeI酶切位点的引物,用RT-PCR方法从东方山羊豆幼嫩叶片总RNA中扩增出GoMIPS基因的cDNA序列,经纯化后,克隆至pMD19-T载体上,构建pMD-MIPS的中间载体,测序鉴定。然后用NcoI和SpeI限制性酶对含有目的基因的pMD-MIPS和pCAMBIA1302空载体进行双酶切,通过酶切鉴定和测序分析表明,已成功构建了CaMV35S启动子驱动GFP报告基因的双元植物表达载体pCAMBIA1302-MIPS。
Abstract
A pair of primers containing the restriction sites for NcoI/SpeI were designed according to the cloned GoMIPS (inositol-1 - phosphate synthase) gene sequence of Galega orientalis. The GoMIPS cDNA was amplified by RT-PCR from the total RNA of the young leaves of the Oriental Galega. After purification, RT-PCR products were cloned to the pMD19-T vector were dealt to construct the pMD-MIPS in the middle of the carrier DNA sequencing. pMD-MIPS containing the target gene and pCAMBIA1302 empty vector with NcoI / SpeI restriction enzyme digestion,the restriction endonuclease and sequence analysis shows that a dual CaMV35S promoter-driven GFP reporter geneplant expression vector pCAMBIA1302-MIPS has been successfully constructed.
关键词
东方山羊豆(Galega.orientalisL.) /
GoMIPS /
pMD19-T /
pCAMBIA1302
{{custom_keyword}} /
Key words
Galega. orientalis L. /
GoMIPS (inositol -1- phosphate synthase) /
pMD19-T vector /
pCAMBIA1302 vector
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1]Toker A. The synthesis and cellular roles of phosphatidylinositol 4,5bisphosphate[J].Curr Opin Cell Biol,1998, 10(2):254261.
[2]Munnikt, Irvine R F, Musgrave A. Phospholipid signaling in plants[J].Biochimica et Biophysica Acta,1998, 1389(3):222272.〖JP〗
[3]Majumder A L, Johnson M D, Henry S A. 1Lmyoinositol1phosphate synthase[J]. Biochimica et Biophysica Acta, 1997,1348(12): 245256.
[4]Bachhawat N,Mande S C. Complex evolution of the inositol1phosphate synthase gene among archaea and eubacteria[J].Trends Genet, 2000,16(3):111113.
[5]Lackey K H, Pope P M, Johnson M D. Expression of 1Lmyo inositol1 phosphate synthase in organelles[J]. Plant Physiol, 2003,132(4): 2 2402 247.
[6]Donahue J L, Alford S R, Torabinejad J, et al. The Arabidopsis thaliana Myoinositol 1phosphate synthase1 gene is required for Myoinositol synthesis and suppression of cell death[J].Plant Cell,2010,22(3):888903.
[7]Hao Chen, Liming Xiong. Myoinositol1phosphate synthase is required for polar auxin transport and organ development [J]. Biol Chem, 2010,285(31): 24 23824 247.
[8]Johnson M D, Sussex I M. 1Lmyoinositol 1phosphate synthase from Arabidopsis thaliana[J].Plant Physiol, 1995,107(2): 613619.
[9]Abreu E F M, Aragao F J L. Isolation and characterization of a myoinositol1phosphate synthase gene from yellow passion fruit (Passiflora edulis f. flavicarpa) expressed during seed development and environmental stress[J]. Annals of Botany, 2007,99(2): 285292.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
山西省农业科学院畜牧兽医研究所基金项(YGG0853)
{{custom_fund}}