Studies On Improvement Of Zhanhua Winter Jujube (Ziziphus Jujuba Mill.) By Genetic Engineering

Zhanhua winter jujube (Ziziphus jujuba Mill.) is an elite variety of winter jujube, a serotinous species of Chinese origin. Zhanhua winter jujube is not only of high nutrition, but also of great economic value. However, some horticultural characteristics of Zhanhua winter jujube, such as the short fruit maturity and storage period, need to be improved. As one of non-climacteric species, the fruit ripening of Zhanhua winter jujube is triggered by elevated abscisic acid level in fruit. Thus, it is possible to delay the fruit ripening and prolong the fruit storage by reducing the synthesis of abscisic acid via antisense strategies. To this aim, we constructed the plant expression vectors carrying the key genes of ABA synthesis, and established the efficient genetic transformation system of Zhanhua winter jujube.Zeaxanthin epoxidase (ZEP) gene and 9-cis-epoxycarotenoid dioxygenase (NCED) gene, the key genes of ABA synthesis, were cloned from salt cress (Thellungiella halophila) by screening the cDNA library. Four plant expression vectors were constructed: pCAMBIA1300-ZEP(+)-als, pCAMBIA1300-ZEP(-)-als, pCAMBIA1300-NCED(+)-als and pCAMBIA1300-NCED(-)-als. Agrobacterium tumefaciens strain LBA4404 carrying the above vectors was used to transform the tobacco leaf discs. Putative transgenic plantlets were analyzed by PCR, and some of transgenic plantlets were confirmed by southern blotting. The transgenic tobacco lines will be used to analyze the function of ZEP and NCED.Shoot tips of Zhanhua winter jujube were used as the transformation receptors and were inoculated with the Agrobacterium LBA4404 strain carrying the vector pCAMBIA1300-betA-als. Some factors influencing the Agrobacterium-mediated genetic transformation were investigated, including concentration of Agrobacterium suspension, inoculation time, cocultivation time and vacuum infiltration. The shoottips were inoculated with OD6oo0.6-0.8 LBA4404 for 10-15min under 50KPa pressure, and then cocultivated 3 to 4 days. The highest efficiency (5.2%) was observed under the above conditions. The presence of betA gene and als gene in putative transgenic plantlets were identified by PCR and PCR-southern blotting. The primers flanking non-T-DNA sequence of pCAMBIA1300-6efA-a/.y were designed to detect whether there was the contamination of Agrobacterium or not. In order to confirm the efficiency of the transformation system, the pCAMBIA1301 vector carrying a gus reporter gene was used under the above optimum conditions. The P-glucuronidase histochemical assays indicated that gus gene can be expressed in the apical meristems of shoot tips of Zhanhua winter jujube.