Establishment Of Narcissus Transformation And Isolation Of Its Agamous Bomologuous Genes And Identification And Analysis Of Brassinosteroid Responsive Genes In Arabidopsis

Chinese narcissus (Narcissus tazetta var Chinensis), blooming during ChineseSpring Festive, is a Chinese traditional flower with high economic value. It ischaracterized by normal vegetative growth under lower temperature, death of leafand differential of big stem in large stem under higher temperature. Only a fewresearches about its micropropagation and radiation breeding were reported.In order to modify the traits of narcissus flower such as form and color by geneengineering, the expressional plasmid of the objective genes and regenerate systemfrom callus after genes transformation are two key steps. It is found that Narcissusexplants should be treated at low temperature (4℃) for 40-60 days, otherwise theexplants will turn to brownish after inoculating on the medium. The contaminatingrate will increase if the low temperature treatment is longer than 60 days.After inoculating 2700 explants on 8 different media, it is found that floral is thebest explant to induce callus on MS medium supplemented with 6-BA0.1mg/1+2,4D1.0mg/1+ NAA0.2mg/1, the best subculture medium is MS mediumsupplemented with 6-BA0.1mg/1+2,4D0.5mg/1+NAA0.2mg/1,and tubelet can beformed on the differential medium supplemented with 6-BA1 .0mg/1+2,4D0.05mg/1+NAA0.2mg/1. In order to establish a genetic transformation system, Hygromycin wasused as selection pressure; Calli became brownish and eventually die on themedium containing 20mg/1 Hygromycin, based on which, a narcissus genetictransformation system was established.To modify narcissus flower organ identity to increase ornamental value, itsAgamous homologous gene was isolated. Agamous gene of Arabidopsis thianiaobtained by RT-PCR was used as a probe for narcissus southern blot. No band wasdetected at high stringency. However 2- 4 bands were found on the filter at lowstringency (hybridized at 42'C, with 25% fOrmenmide, washed with2xSSC+0.1%SDS, lxSSC+0.l%SDS at 42'C). High degenerate nest primers werethen designed according to the Agam ous consensus sequence. 3 bands of 350bp,500bp and 750bp were amPlified by RT-PCR. The PCR products of 350bp and750bp were sequenced and blasted against GeneBank. The sequence of 350bp PCRproduct was found to be highly similar to the Agamous gene of other plants. In orderto clone the full length of the cDNA, nest primer was designed according to thepatial cDNA sequence. 3 bands were amPlified by 5'-RACE and 2 band wereaxnplified by 3'-RACE. The 750bp 5'-RACE product and the 500bp of 3'-RACEproduct were sequence and blasted against GeneBank.. The result shows that thesetWo sequences were similar to other plant Agamous genes of different plants and theyhave 200bp over1aP to each other. Therefore a full length cDNA sequence wasobtained. This cDNA is 1320bp long and has an open reading frame encoding 230amino acids residues. The putative amino acid sequence of conserved aI, aII, 6I, 6IIdomain are identical to that of other plant. The cloned narcissus Agamous gene lays asolid to further genetic engineering.Brassinolide (BL) is a new tyPe of plan hormone which plays multiple roles inplat cell elongation and division, plant reProduction, and photosynthesis. BL alsoincreases seedling grOwth, biomass and stress tolerance. Although BL have broadphysiologica1 function, its molecular mechanism is still unclear. In order to identifyand analysis BL responsive genes and further understand its molecular mechanismthat regu1ated the p1am groWth and development, 5l BL responsive genes wereidentified from l3,000 cDNA clones by cDNA array. 51 clones were sequenced andblasted against GeneBank database. Four tyPes of differeniial expression genes wereobtained. They are genes related to cell division, signal tTansduction, stress and novelgenes. The three genes (CycD3 MyB-like and Mto2 ) related to cell division werestudied in detail.