Preliminary Study On Construction And Transformation Of RNAi Expression Vector Targeting ACO Gene In Tulipa Gesneriana

As one of the major contributors to the cut-flower market and garden greening, Tulip (Tulipa gesneriana L.)is widely favored by people all over the world. But like other fresh cut flowers, tulip will decay rapidly while blooming. How to prolong the florescence has become one of the main subjects for studying recently. In this study, ACC oxidase (ACO) gene fragment was cloned from the petals of tulip. The ihpRNA structures of plant RNAi expression vectors of ACO gene were constructed and integrated into tulip tissue culture plants, and controlling the senescence of Tulip by manipulating the expression of ethylene, the purpose of prolonging the florescence of tulip can be attained. Three programs were done.1. Cloning of ACC oxidase gene of tulip and constructions of its plant RNAi expression vectors.A pair of primers was designed according to the 1-aminocyclopropone-1-carboxylic acid (ACC) oxidase gene sequence of tulip, and the primers were used to amplify the genomic DNA fragment by polymerase chain reaction (PCR) by taking cDNA from petals of tulip. The PCR product was cloned into pUCm-T vector. Sequencing indicated that the ACC oxidase gene fragment was successfully cloned. The ACO gene fragment was linked sense and antisense to the pBSin plasmid, between the sense and antisense gene sequence was the intron, so the hpRNA structures were constructed. Then, the hpRNA structures were cutted from the pBSin plasmid, and linked to the pCAMBIA35S-1301 binary vectors, the RNAi expression vectors of ACO gene were constructed.2. Study on tissue culture of Tulipa gesneriana L.To establish an regeneration system for tulip in vitro, the interfoyles, cephalophorum, flower stems, petals, thrumbs, and pistils of tulip were used as explants. The influences of plant growth regulators on bulblets sprouting were research. As a result, the optimum mediums of every step are screened. As explants, the differentiation ability of the flower stems, cephalophorum, and pistils were excelled than interfoyles and others. The flower stems, cephalophorum, and pistils all can be used for inducing callus and buds. The medium MS+0.5 mg/L 2,4-D was propitious to induction and proliferation of callus, the medium MS+0.2 mg/L 2,4-D+6.0 mg/L ZT was propitious to induction and proliferation of buds and differentiation of callus. On rooting culture, the 1/2MS+1.0 mg/L NAA+2.0 g/L A.C is the optimum medium for rooting culture.3. Transformation of tulip with the RNAi expression vectors of ACO geneThe RNAi expression vectors of ACO gene were transformed into A.tumefaciens GV3101, A.tumefaciens strain GV3101 harboring hpRNA structures of ACO gene RNAi expression vectors, and they were used to transform tulip buds and callus.