Genetic Transformation Of ZDS Antisense Gene In Narcissus Tazetta Var. Chinensis

Narcissus tazetta L. var. chinensis is perennial single cotyledon herbaceous plant belongs to Narcissi genera, Amaryllidaceae family which has become one of the traditional famous flowers enjoyed by people, for the flowers of ornamental value and economic value. In the experiment, the reel bulbs of Narcissus tazetta L var. chinensis were used as test materials to induce bulbs, establishing efficient in vitro regeneration system of Narcissus tazetta L. var. chinensis. The ZDS antisense gene plant expression vector has been built according to the known ZDS gene sequence. And adopting the method of agrobacterium mediated root carcinoma to transform the ZDS antisense gene into Narcissus tazetta L. var. chinensi. The main results are as follows:1 The establishment of the stable and efficient regeneration system of Narcissus tazetta L. var. chinensisThe results showed that the effect of disinfection in the bulbs with 55 ℃ water treatment 12 min 5 min+ 0.1% mercuric chloride was very well while the raw materials was three years bulbs of Narcissus tazetta L. var. chinensis. Using 6-BA, NAA and 2,4-Das factors to process orthogonal design test to induce bulbs, the result was that the best of bulbs induction culture medium was modified MS medium+2.0 mg/L6-BA+0.0 mg/L2, 4-D+0.6 mg/L NAA with the induced rate of 214.10%. And adopting orthogonal design which used the 6-BAn NAA and sugar concentration as factors effect on the small bulbs receptor system, the results showed that bulbs induction rate of 88.89%cultivating modified MS medium+2.0 mg/L6-BA+1.0 mg/L NAA+30 g/L sugar medium. The best breed rooting medium of small bulb was 1/2 MS+0.2 mg/L NAA with the rooting rate of 87.00% while taking peat soil, a mixture of perlite and soil matrix for transplanting substrate, with the transplanting survival rate of 95.00%.2 Construction of ZDS antisense gene carrier of Narcissus tazetta L. var. chinensisUsing the flower petals and deputy crowns of Narcissus tazetta L. var. chinensis as materials to extract total RNA and reverse transcribe synthesize the first chain of cDNA. According to ZDS cloning gene sequences of Narcissus tazetta L. var. chinensis that has been cloned and pCAMBIA1301 double enzyme sites GUS carrier, we designed a pair of specific primers which consisted of Sad and XbaI restriction enzyme sites. Narcissus tazetta L. var. chinensis ZDS gene was obtained by RT-PCR, recycling purposes fragments, then connecting the purpose fragments and PMD18-T carrier, transforming, and obtaining the recombinant plasmid finally. Then conducting double enzyme reaction with pCAMBIA1301 double GUS carrier, connecting directly recycle products, inserting reversely complement the cloned fragment to pCAMBIA1301 double GUS plant expression vector, obtaining recombinant plasmid P1301-ZDS, after that plasmiding the recombinant into root cancer Agrobacterium, and PCR detecting, the results showed that successful build plant antisense vector.3 The establishment of Root carcinoma agrobacterium mediating small bulbs of Narcissus tazetta L. var. chinensis transformation systemThe test showed that using this method as follows could process stable conversion test. Preincubating 6d before infection, using MS liquid medium as the root of carcinoma agrobacterium suspension and making the root agrobacterium suspension concentration be o.6, the sugar concentration in the root agrobacterium suspension be 60g/L, the time of root carcinoma agrobacterium infecting bulb be 30min, and co-culturing in the medium whose pH was 5.8 in 25 ℃ in the dark conditions.4 Screening of resistance bulbs and detection of regeneration plantsAntagonistic bulb screening test showed that the effect of delay screening method was the best.12 strains of intense hygromycin resistant plants were obtained through the delay screening. Conducting GUS histochemical detection of blades in the 12 strains of China narcissus transgenic regenerated plants found that 10 of the 12 strains of Narcissus tazetta L. var. chinensis transgenic plants were stable expression of GUS gene. And PCR detection of transgenic regeneration plants after hygromycin marker genes. the results showed that 11 strains of transgenic plants were detected hygromycin gene. It showed that exogenous hygromycin gene had integrated into the genome of Narcissus tazetta L. var. chinensis in the 11 strains of transgenic plants. Contrasting the transformed plants and the non-transformed plants, the phenotypes were indifference. (12 strains of transgenic plants which were transplanted to fields can survive all.)In a word, efficient Narcissus tazetta L. var. chinensis regeneration system was established in this experiment, antisense gene plant expression vector was constructed successfully and transformation conditions was optimized in the root carcinoma agrobacterium mediating transformation of Narcissus tazetta L. var. chinensis bulb. In the experiment, we transferred ZDS antisense gene into the small bulbs of Narcissus tazetta L. var. chinensis for the first time, explored the method of screening of resistance bulbs of Narcissus tazetta L. var. chinensis and obtained the transgenic plants successfully to provide referential basis and technology platforms for the genetic transformation of Narcissus tazetta L. var. chinensis.