Identification Of Three PttKN1-transgenic Plants And Analysis Of Pigment-altered Lines

The shoot development of plant depends on the perpetuation of a group of undifferentiated cells in the shoot apical meristem (SAM). KNOX genes encode homeodomain-containing transcription factors that are essencial for meristem maintenance and proper patterning of organ initiation. PttKNl (P.tremula×tremuloides KNOTTED1), isolated from the vascular cambial region of a hybrid aspen, belongs to KNOX I family. It has been induced in to Arabidopsis, tobacco, Petunia, Begonia, Beta vulgaris var cicla, Lotus corniculatus L., Coleusbl blumei, Dianthus caryophyllus L. and Celosia cristata L. in our previous work. Three species of the transgenic plants were choosed for further study in this paper, which were PttKN1-transgenic tobacco, Coleus blumei and Beta vulgaris var cicla. They were analyzed at the level of morphology, physiology and molecular biology. The main results were showed as follows:1. PCR, RT-PCR and PCR-southern blotting were performed. Three pairs of primers were used in PCR analysis, which were designed in the sequences of PttKN1, NPT-II and 35S-promoter separately. Two different hybridization probes were applied in PCR-southern blotting, which were designed based on the sequences of PttKN1 and NPT-II. Positive bands indicated that the PttKN1 was integrated into the genome of the three plants; the phenotypic variation should be caused by the insertion of exotic gene.2. Compared with wild type plants (WT), both the transgenic Beta vulgaris var cicla and Coleus blumei showed color-altered phenotype. Several pigment-related physiological and biochemical analysis were performed. The content of beet red pigment in transgenic green-type Beta vulgaris var cicla (GT) was much lower than WT; the green-type plants showed little red pigment content. Similar result was observed in the content of anthocyanin in transgenic Coleus blumei, which contained much less anthocyanin compared with WT.3. The tyrosinase activity in Beta vulgaris var cicla, which was a limiting factor in the synthesis of red beet pigment, was detected 3.7 times higher in wild type than transgenic green line. Similarly, In Coleus blumei, the soluble sugar content in WT was found much higher than transgenic green-type line. These results implied one of the factors causing color-altered phenotype:PttKN1 might interact with other transcriptional factors which lay on the upstream of the synthesis way of both red beet pigment and anthocyanin.