Genetic Transformation And Functional Analysis Of Pttkn1 Gene In Nicotiana Tabaccum L.

A series of phenotypical alterations in plant architecture, leaf shape and flower pattern, with the abnormal vascular tissues, had been observed, when the KNOX I gene originating from herbaceous plants expressed in plants. Few studies concerning the functions of KNOX I gene isolated from woody plant had been reported. Pttkn1 (Populus tremula×tremuloides KNOTTED1) gene is a new KNOX gene that was isolated from the vascular cambial region of hybrid aspen. Sequence alignment of Pttkn1 gene to a number of KNOX genes showed that it belonged to class I KNOX genes encoding transcription factors, which took an important role in regulation of plant growth and development. To investigate the effects of Pttkn1 gene on plant morphogenesis and development of vascular tissue, Pttkn1gene had been introduced into the genomes of Nicotiana tabacum L. using the leaf disc method mediated by Agrobacterium tumefaciens. A series of novel phenotypes obviously different from the wild type were observed in the transgenic tobacco plants. The major results were as bellow:1. When Pttkn1 gene expressed in Nicotiana tabacum L., plants resistant to Kanamycin exhibited typical phenotypical features, 1) The changes of the leaf types, including shortened petiole, anisomerous leaves, lobed leaves, wavy leaves, the abnormal location of the petiole, petioleless leaves, and formation of the ectopic meristems on leaves; 2) The changes of the plant architecture, including loss of apical dominance, and the scrubby and dwarf plants; 3) The configuration, the color and the blooming time of the flowers were changed; 4) The color of the seed capsules had changed into dark brown from light brown.2. RT-PCR analysis indicated that Pttkn1 gene strongly expressed in the plants resistant to Kanamycin, but not in wild-type Nicotiana tabacum L., indicating that the gene had been successfully introduced into the plants. There is certain relevance between the expressional intensity of the gene and the phenotype order of severity.3. Analysis of vascular bundle in leaves from transgenic plants showed that venation patterns of leaves had changed, when they were compared to wild type. 1) The midvein shortened and broadened; 2) Midvein was not obvious. 3) Two or more midvein appeared in a leaf; 4) Petiole formed on the midvein; 5) The lateral veins emanated irregularly from midvein; 6) The number of lateral veins emanated from midvein were dicreased and formed a closed loop in the middle of the leaf.4. The anatomical structure of transgenic plants showed that ectopic expression of Pttkn1 gene leaded to alterations in cell morphology of leaves and rachis compared to wild type, including: the cell shapes were irregular; leaves contain unrecognizable palisade parenchyma layer; mesophyll cells packed tightly and increased in number, the shape of rachis alterated, the morphology of vascular bundles and their structures changed. Ectopic meristem initiated on the leaves and petioles.5. Analysis of the scanning electron microscope indicated that the epidermal cell shapes of the transgenic plants had changed into smaller irregular cells. The density of the epidermal hair is bigger and abnormal compared with wild type.The results of our experiments suggest that Pttkn1 gene plays an important role in regulating the formation and maintenance of shoot apical meristem and the development of vascular tissue. And the gene as a transcript factor may regulate a number of target genes which are related to development of shoot apical meristem and vascular tissue.