Cloning And Expression Analysis Of GME And ACT Gene From Caragana Korshinskii And Construction Of Plant Expression Vector

A pair of degenerate primer was designed according to the conserved sequence of GME from other plants. A GME gene named CkGME and an ACT gene named CkACT were separated from Caragana korshinskii by using the RT-PCR and RACE technology. The bio-information analysis by using DNAMAN, DNAStar, Blast P or physical and chemical property analysis of CkGME and CkACT showed the following results:1,The full-length cDNA of CkGME (accession number FJ603689) is 1604bp and contain an 1134bp ORF which coding 377 amino acids. The relative molecular weight is 4.2 622×104 and molecular formula is C1853H2921N491O560S20. 5'-non-coding region is 351bp and 3'-non-coding region is 94bp.2,The full-length cDNA of CkACT(accession number is FJ485727) is 1607bp and contain an 1134bp ORF which coding 377 amino acids. The relative molecular weight is 4.1679×104 and molecular formula is C1853H2921N491O560S20. 5'-non-coding region is 151bp and 3'-non-coding region is 293bp.3,The homology of CkGME compared with GME of Zea mays,Arabidopsis Thaliana,Glycine max,Medicago truncatula,Malpighia glabra,Oryza sativa japonica Group,Solanum tuberosum,Vitis vinifera is 92%,90%,91%,92%,91%,95%,93% and 91% respectively. The number of amino acid residue of all species is between 376 to 378 and all parts of region is conserved except the N-terminator region.4,The homology of CkACT compared with ACT of Zea mays,Oryza sativa,Avena nuda,Arabidopsis thaliana,Nicotiana tabacum,Vigna radiata,Pisum sativum,Cicer arietinum,Medicago truncatula and Trifolium pretense is 97%,96%,96%,96%,97%,98%,97%,96%,99% and 95% respectively.5,The CkGME is the unstable protein with protein unstable index of 40.47 and the CkACT is the stable protein with protein unstable index of 35.02.6,The semi-quantity RT-PCR analysis of the expression of CkGME with ACT as a internal reference gene showed that the expression had no difference compared with control after treated at 4℃for a during time, but was down-regulated after treated with GA3.7,The coding region amplified by PCR is used to construct the pCAMBIA1300-GME recombinant plasmid which is converted to the Agrobactrium tumefaciens.The result of PCR and BamHⅠ/SalⅠdouble digestion showed that the plant over-expression vector had been constructed successfully.