Construction Of The Vector For Endosperm Specific Polyphenol Oxidase RNA Interference And Its Transformation In Common Wheat

Polyphenol oxidase (PPO) is one of the primary factors in brown causing of the dough. Blocking or limiting the express of ppo genes is meaningful to enhance whiteness of flour product. The establishment of a practicable agrobacterium mediated transformation system is the key technology to make the break through in wheat transformation. The present study comprised two experiments: one is to development of high efficiency agrobacterium transformation method; the other one is the construction of the ppo RNAi vector and transfer it into wheat.1. The development of the high efficiency agrobacterium-mediated transformation method. In the present experiment, mature embryos were used as the explants with the advantage of obtaining conveniently. The results indicated that by the injection method, the average frequency of positive plants with gus expression was 78.1%, while average frequency of the positive gus signal in spikes was 73.2%. Whereas, by needle puncture, average frequency of the positive plants was 59.8%, in which the average frequency of the positive gus signal appeared in spikes was 53.6%. The average transformation frequency in cultivar K35 was 74.2%, in which the aveage frequency of the positive spikes with gus expression was 53.6%. While average transformation frequency in cultivar Jinyi No. 1 was 65.6%, and in which the aveage frequency of the positive spikes with gus expression was 76.9%. When the transformed seeds were soaked in water for only four hours with no shoots, the frequency of the positive gus expression in plantlets was 67.7%, in which the aveage frequency of the positive spikes with gus expression was 63.6%. And when the seeds with no shoot which were obtained after soaked in water 4 hours and left overnight at 22℃, the frequency of the positive gus expression in plantlets was 69.7%, in which the aveage frequency of the positive spikes with gus expression was 65.2%. when the seeds with 1mm shoot which were obtained after soaked in water 4 hours and left two days at 22℃, the frequency of the positive gus expression in plantlets was 72.1%, with the frequency of the positive spikes was 65.9%. So, compared with K35, Jiyin No.1 is the preferable cultivar transfomed by injection the seeds with 1mm shoot tip.2. Vector construction. The third exon was chosen as the target sequence. The first step is to construct a mediated vector pZlbx-ppoRNAi which contains embryo specific promoter Bx17 and ppo specific RNAi cassette. By using proper restriction sites, mediated pBluippo and pBluippopBIOS were constructed and then transferred the whole cassette to pBIOS1952 which contains Ac/Ds transposon system. Finally, the expression vector piPPO used for agrobacterium mediated transormation were constructed which contains Ac/Ds marker deletion system.3. Transform piPPO into wheat. Five hundreds of immature embryos were isolated after inoculation with agrobactium EHA105 which contained the constructed plasmid of piPPO. After the callus induction and regeneration with Slection agent of G418, ten survived plants were obtained. Among them, 5 positives were confirmed by PCR amplification.