Cloning and bioinformatics analysis of Actin gene in Fritillaria thunbergii / 中成药

ABSTRACT

AIM To clone the Actin gene in Fritillaria thunbergii Miq.and to make bioinformatics analysis.METHODS The total mRNA in roots,stems,leaves,flowers and bulbs of F.thunbergii was extracted,and the degenerate primer was designed and synthesized.With total mRNA in leaves as a template,the conserved fragments of Actin gene was cloned by RT-PCR and Ta cloning technology.Using this gene as a reference gene,tissue specificity expression analysis was adopted in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene.RESULTS One gene sequence (463 bp) was obtained by RT-PCR amplification and Ta cloning.The Actin gene in F.thunbergii showed high similarities to those in Lilium regale Wilson,Tulipa gesneriana,Ornithogalum caudatum Jacq.,Dendrobium officinale Kimura et Migo,Diospyros kaki Thunb.,Betula luminifera H.Winkl.and Zea mays L.(84％-98％),the homologies of its amino acid sequence to Drosera adelae F.Muell,Brassica napus L.,Vanilla peanigoeia Ancer,L.regale,Jatropha carcas L.,Lycium barbarum L.and Rhizophora stylosa amino acid sequences were all more than 89％,and the Actin protein had close genetic relationships with Lotus corniculatus L.,L.regale and T.gesneriana.The expressions of HMGR gene in various parts of this plant showed obvious differences,which was in sequence of bulbs ＞ flowers ＞ leaves ＞ stems ＞ roots.CONCLUSION It is the first time that Actin gene (named as FtActin) is coloned in F.thunbergii,which can lay the basis for its effective application.