Cloning Of Dihydroflavonol4-Reductase Gene CDNA Fragment From Loropetalum Chinense Var. Rubrum And Construction Of RNAi Vector And Tobacco Transgenic Research

Loropetalum chinense var.rubrum belongs to the Hamamelidaceae, Hamamelidoideae rubrum species, which has been widely used as Colored-leaf plants because of its brilliant and colorful leaves, Strong adapt ability, stress tolerance and plasticity, easy breeding, in the area south of the Yangtze River. Leaves are the main viewing parts, leaf color is to determine the main factors of its ornamental value, but in the summer of Changsha area prone to "turn green". Although some scholars have done some research on the physiological and biochemical mechanisms, but still failed to explain the fundamental mechanism of coloration. Dihydroflavonol4-reductase (DFR) is the main enzyme in anthocyanin synthesis and played a key role in the formation of the color of ornamental plants. In this study, L. chinense var.rubrum cultivars’Dayehong’was used as material, DFR gene cDNA fragments were cloned, constructed RNAi vectors and infection by Agrobacterium into the tobacco. In order to reveal the role of DFR gene rubrum Leaves change color in the process of laying the foundation. The main results are as follows:1. DFR gene cDNA fragments were cloned from the young leaves of the L. chinense var.rubrum cultivars’Dayehong’,which is395bp, encoding131amino acids, named " Lcvr DFR1". By comparing its sequence with homologues from other plants by BLASTn, found a large DFR sequence homology of the sequence and the same source logged Paeonia suffruticos, Lotus japonicas, Fragaria vesca subsp, Vesca bifunctional, Lycium barbarum DFR sequence about80%. The amino acid sequence of the deduced amino acid sequence had the same highly homologous with amino acids encoded by DFR of Populus trichocarpa, Malus domestica, Vitis rotundifolia, Pyrus pyrifolia, Crataegus monogyna, Prunus avium, Rubus hybrid cultivar, Vitis bellula were100%coverage of the amino acid sequence homology of about85%.2. RNA interference vector of I. chinense var.rubrum DFR gene was constructed. The two LcvrDFR1sequence, forward and reverse connected at both ends of the GUS gene, which could form hair-spin constructs, as a bridge to pBin438expression vector was constructed successfully with antibiotics (Kan) Select the GUS gene marker gene Lcvr DFR1-RNAi expression vector, and transferred it into the Agrobacterium tumefaciens strain EHA105by freeze-thaw method, and gained expression strain. 3. Tobacco aseptic leaf was used as explants, and the gene was successfully transferred into tobacco by Agrobacterium tumefaciens strain EHA105harboring the expression vector pBin438and co-cultured for3days in darkness on the culture medium (MS+6-BA1.0mg/L+NAA0.5mg/L). Explants were then transferred onto the Selection medium containing antibiotics50mg/L Kan and200mg/L Cef. Small leaves of adventitious shoots differentiated from explants were cut out and identified by PCR amplifying. The results showed that the RNAi elements of LcvrDFR1had been integrated into the genome of tobacco.