Construction Of SSH-cDNA Library Of Chinese Jujube Infected With Phytoplasma And Its ESTs Analysis

Chinese jujube is one of the most representative fruit tree in China. Jujube witches-broom disease (JWB) caused by phytoplasma is the deadly infectious disease of Chinese jujube and the majority of Chinese jujube cultivars are highly sensitive to JWB. In this experiment, the scions of cultivar ’Xingguang’ with high resistance to JWB were grafted onto the diseased and healthy trees of sensitive cultivar ’Zanhuangdazao’, respectively. A SSH (suppression subtractive hybridization) library of Chinese jujube under stress of JWB was constructed and the differential fragments in the library were sequenced and analyzed, The aim of this study is to obtain genes related to the resistance to JWB and lay a foundation for revealing the molecular mechanism of JWB resistance and the follow-up molecular resistance breeding in Chinese jujube. The main results were as follows:1. The jujube SSH library under phytoplasma stress was constructed successfully.1300clones were preserved in the library, and500clones were selected randomly and analyzed. There were470positive clones (94%) with one band,19positive clones (3.8%) with two bands and11empty vectors (2.2%) in the library. The inserted fragments of the library were between200bp and1000bp, which with an average length of500bp.2.470positive clones were sequenced and200non repetitive ESTs were got. BLASTn and BLASTp results revealed that89cDNAs had signicant sequence homologies with known sequences in the NCBI database, which accounting for45.5%of all the number of ESTs;102cDNAs had signicant sequence homologies with unknown sequences, which accounting for51%; and9cDNAs were not similar sequences or proteins, which accounting for4.5%.3.89expressed ESTs of known function under phytoplasma stress were classifed. The identied cDNAs encoded proteins involved in cellular processes such as:resistance/defense, transport, signal transduction pathway, metabolism, protein synthesis and processing and cell growth/split genes. Among them, resistance/defense genes were account for the largest proportion (23.6%), and then followed by transport and signal transduction pathway genes.4.10ESTs related disease resistance were selected and carried out RT-PCR analysis. The results showed that the expression of these genes in ’Xingguang’ of the early stage(40d after grafted), middle stage (60d and75d) and later stage (95d,115d) displayed a tendency of low-high-low. In the middle stage, the content of phytoplasma was the peak, and the expressions of these genes were also the highest. The expression change of those genes was consistent with the changes of pathogen and phenotypy.