Genetic Transformation Of PttKN1 Gene And Analysis Of Its Functions In Lotus Corniculatus

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 to a number of plant KNOX genes showed that it belongs to class I knox genes. A high efficient regeneration system of legume forage Lotus corniculatus L. was established, and PttKN1 gene had been introduced into Lotus corniculatus by Agrobacterium and its functions had been studied from several aspects in this study. The major results were as bellow:1. A high efficient regeneration system of Lotus corniculatus was established. Shoots were obtained from cotyledon and hypocotyl explants and the rate of bud differentiation were 82.0% and 96.0% respectively. And their rooting ratio was 82.9% on MS medium. The plantlets were transplanted in different kinds of media: 100% perlite, 100% vermiculite, 100% peat soil and their mixture soil of perlite: vermiculite: peat soil (1:1:1). It was found that the 100% vermiculite was better than others, and the survival rate was 94.1%.2. RT-PCR analysis indicated that wild-type Lotus corniculatus plant did not expressPttKN1 gene but transgenic plants resistant to kanamucin expressed it.3. When the PttKN1 gene was overexpressed in Lotus corniculatus, the morphology of transgenic plants was altered: 1) The complexity of the leaves in Lotus corniculatus was increased, including increased leaflet initiation, four to five four to five leaflets in a rachis. 2) Abnormal compound leaves and reduced leaflet were observed; The terately compound leaf of Lotus corniculatus became simple leaf or two leaflets in a rachis, which always accompanied with normal stipules. 3) Lobed leaves formed, or the number of the leaflet were normal but the symmetry of leaves were changed and some leaflets were smaller than others.4. Transgenic plant always associated with defects in internode elongation and growth retardation which resulting in dwarfed, acervate plants; Some of the stems lost shoot apical meristem and lead to the plants could not continue to grow.5. Overexpression of PttKN1 gene lead to a disordered venation. 1) Two or more midvein appeared in a leaflet. 2) Lateral veins emanated from midvein were lessened and formed a closed loop in the middle of the leaflet. 3) A loop of vascular bundle was formed in the petiole.6. The anatomical structure of transgenic plants showed that ectopic expression of PttKN1 lead to an altered pattern of cell morphology in leaflets and rachis, including: the cells shape were irregular; leaves did not contain a recognizable palisade parenchyma layer; mesophyll cells packed tightly and increased at least two cell layers; collenchyma in the rachis disappeared and the shape of rachis was changed from triangular into round or oval as well as the cells in the rachis; the number of vascular bundles decreased and their structure were simplified. Ectopic meristem initiated in the center of the petiole.Taken together, PttKN1 transformation caused the functional alterations of genes in the transgenic plants, and further affected a series of changes in morphology, structure and development, which reveals that PttKN1 gene, a transcript factor, may regulate a number of genes which are involved in many aspects of plant development.