Articles
OVER-EXPRESSION AND SILENCING OF KXHKN5 GENE IN KALANCHOE × HOUGHTONII
Article number
836_38
Pages
265 – 269
Language
English
Abstract
K. × houghtonii (n=51), a triploid interspecific hybrid between K. daigremontiana Hamet & Perrier (n=17) and K. delagoensis Ecklon & Zeyher (n=34), forms epiphyllous buds on leaf margin notches in response to a long day photoperiod (vegetative vivipary). Several well known class 1 KNOX genes, as Kn1 from maize, STM, KNAT1 and KNAT2 from A. thaliana and Bkn3 from barley, play an important role in meristem formation and maintenance.
In an attempt to characterize KNOX genes involved in vegetative vivipary in K. × houghtonii, over-expression and post-transcriptional gene silencing (PTGS) experiments were performed employing KxhKN5 gene (EU240661). To accomplish over-expression, the complete cDNA sequence of the gene (1161 bp), overdrive by 35S promoter and NOS terminator, was cloned in the binary vector pGreen II NPTII. The PTGS construct was prepared by cloning in pJM007 (Schattat et al., 2004) a 326 bp fragment of the gene in sense and antisense orientation in the specific cloning sites located at the left and at the right of the PIV2 intron; silencing cassette was excised from pJM007 and cloned into the binary vector pGreen II NPTII; the derived vectors were then transferred to A. tumefaciens. Genetic transformation and selection on medium containing kanamycin as selective agent were conducted.
To localize KxhKN5 mRNA in meristems of K. × houghtonii, in situ hybridization was performed with a digoxigenin (DIG)-labelled RNA probe according to the DIG RNA labelling KIT, with minor modifications.
Over-expression and PTGS of KxhKN5 affect plant architecture and leaf shape as reported for class 1 KNOX genes cloned from other species.
Ectopic buds and shoots formation was not observed in transgenic plants over-expressing KxhKN5; furthermore the vegetative vivipary was reduced in the transgenic clones with extremely modified phenotype likely as a consequence of the leaf shape modification.
In an attempt to characterize KNOX genes involved in vegetative vivipary in K. × houghtonii, over-expression and post-transcriptional gene silencing (PTGS) experiments were performed employing KxhKN5 gene (EU240661). To accomplish over-expression, the complete cDNA sequence of the gene (1161 bp), overdrive by 35S promoter and NOS terminator, was cloned in the binary vector pGreen II NPTII. The PTGS construct was prepared by cloning in pJM007 (Schattat et al., 2004) a 326 bp fragment of the gene in sense and antisense orientation in the specific cloning sites located at the left and at the right of the PIV2 intron; silencing cassette was excised from pJM007 and cloned into the binary vector pGreen II NPTII; the derived vectors were then transferred to A. tumefaciens. Genetic transformation and selection on medium containing kanamycin as selective agent were conducted.
To localize KxhKN5 mRNA in meristems of K. × houghtonii, in situ hybridization was performed with a digoxigenin (DIG)-labelled RNA probe according to the DIG RNA labelling KIT, with minor modifications.
Over-expression and PTGS of KxhKN5 affect plant architecture and leaf shape as reported for class 1 KNOX genes cloned from other species.
Ectopic buds and shoots formation was not observed in transgenic plants over-expressing KxhKN5; furthermore the vegetative vivipary was reduced in the transgenic clones with extremely modified phenotype likely as a consequence of the leaf shape modification.
Publication
Authors
M. Laura, C. Borghi, C. Regis, A. Cassetti, A. Allavena
Keywords
epiphylly, in situ hybridization, Kalanchoe × houghtonii, KNOX genes, PTGS
Online Articles (40)