| Amygdalus Ledebouriana Schlech, is also knowned as wild almond, and belonging to Rosaceae Prunoideae Amygdalus subgenus of Prunus deciduous shrubs, was the relic deciduous tree species of the Miocene epoch of the Tertiary period, and referred to the "living fossil" of the plant kingdom. Because of the wild almond forest pests, and man-made destruction, so it is very important to strengthen the scientific research and protection of wild almond. In this study, plants of five population groups, totally 150 copies of materials, were collected in Yumin, Tacheng, Tori, Burqin, and Habahe of Xinjiang. By homology cloning technology, one genotype of self incompatible relative wild almond gene SSK1 were chosen and cloned, and by bioinformatics software to the cloned gene was analyzed, and then through the construction of Petunia hybrida genetic transformation system, of the function of wild almond self-incompatible gene SSK1 was verified. It is hoped that the research could provide a reference for the protection of wild almond, and future research on the self incompatibility mechanism.1. In this research, the leaves and pollen of wild almonds of Xinjiang area were chosen as the materials. Through the Rt-PCR and biological sequencing technologies, a new full-length SSK1 gene was acquired, and named as Pet SSK1. The results showed that the full-length of Pet SSK1 was 602 bp and the open reading frame was 534 bp, encoding 177 amino acids; through the bioinformatics analysis, the molecular formula of Pet SSK1 protein was C910H1421N233O295S6 and the relative molecular mass of it was 20,538.0, and its estimated half-life was 30 h in theory; moreover, the result showed the hydrophobic index and the grand average of hydropathicity of Pet SSK1 protein are 41.48 and-0.567 respectively, indicating that Pet SSK1 protein belonged to the unstable water-soluble protein. It is the first report that a full-length SSK1 gene has been cloned from wild almonds of Xinjiang area.2. Through the construction and optimization of Petunia hybrida regeneration system, the most suitable method for Petunia seed sterilization is found out: the seeds are dealt with 75% alcohol for 30 s, and 0.1% mercuric chloride for 6min. The most suitable culture medium for inducing the growth of adventitious bud is MS+2.0mg/L 6-BA+0.2 mg/L NAA, and the optimum rooting culture medium is MS+0.4 mg/L IBA. Eventually, by hardening and planting, the well-grown Petunia hybrida seedlings were cultivated.3. The expression plasmid p CAMBIA1303-Pet SSK1 of self-incompatibility related gene Pet SSK1 was constructed. Then it was transferred into Petunia by Agrobacterium tumefaciens mediated method. Besides, the effects of different kinds and concentration of antibiotics on Petunia regeneration were investigated. The results showed that the Petunia hybrida seedlings transformed from self-incompatibility related gene Pet SSK1 of Xinjiang wild almond was gotten successfully. The sensitive concentrations of Kan and Cef were 25 mg/L and 300 mg/L, respectively. And the Petunia were further confirmed as positive plants by PCR detection. |
PDF Full Text Request