Cloning And Expression Of SnRK2 Family Genes In Poncirus Trifoliata And The Interaction With ABF

Trifoliate orange(Poncirus trifoliata(L.) Raf.) is widely used as rootstock in citrus,which is highly tolerant to cold and disease, but sensitive to drought. Improvement of drought tolerance in trifoliate orange is significant for the development of citrus industry.Sn RK2(Sucrose non-fermenting 1-related protein kinases 2) is a class of Ser/Thr proteins that are specific to plant. Studies have shown that a subfamily of ABA-activated Sn RK2 s are positive regulators of ABA signing and play important role in osmotic stress tolerance. The regulation of Sn RK2 and AREB/ABF is important in abiotic stress mediated by ABA signaling. In the present study, we identified and characterized Sn RK2 gene members in sweet orange(Citrus sinensis(L.)Osbeck), and then analyze the interaction between Sn RK2 and ABF2 pathway in trifoliate orange.1. Based on bioinformation analysis, we identified seven Sn RK2 gene members in sweet orange genome. Then we predicted and analyzed physical and chemical characteristics, secondary structure, gene structure, motif, C-terminus sequences and EST.The results showed that the Sn RK2 s identified in sweet orange were hydrophilic proteins.Secondary structure were mainly α-helix, irregular curl and extended strand. The gene structure analysis showed that the exons and introns were conserved, having nine exons.The Sn RK2 s genes in sweet orange were classified into three subfamilies. The motif and C-terminus sequence analysis showed that the sweet orange Sn RK2 s protein kinases were highly conserved. The EST analysis shows that the seven Sn RK2 s were expressed in 10 tissues, mainly in leaf, fruit, flower and callus.2. In order to analyze the function of Sn RK2 s in trifoliate orange, we analyzed the expression patterns of Sn RK2 s genes under drought, ABA stress. The results suggest that trifoliate orange Sn RK2.4 was up-regulated significantly by drought, Sn RK2.3、Sn RK2.6and Sn RK2.10 were up-regulated slightly by drought, while Sn RK2.5 、 Sn RK2.7 and Sn RK2.8 could not be activated by drought. Under ABA treatment, Sn RK2.4 and Sn RK2.5 were up-regulated significantly, Sn RK2.3、Sn RK2.6 and Sn RK2.8 were up-regulated slightly, whereas SnRK2.7 and SnRK2.10 could not be activated by ABA. Sn RK2.4、Sn RK2.3 and Sn RK2.6 were activated by drought and ABA, implying that they may play roles in ABA signaling.3. BIFC assay showed that trifoliate orange Sn RK2 genes could interact with ABF2 in cell nucleus. And we also demonstrate that Sn RK2.3 and Sn RK2.4 were located in plasmalemma and nucleus. Furthermore, the result of prokaryotic expression showed that Sn RK2.4 protein in trifoliate orange could express in BL21 Escherichia coli.