Differential Expression Of Resistance Gene For Jujube Witches Broom

Chinese jujube (Ziziphus jujuba Mill) is important and specific fruit tree in china. Jujube witches broom (JWB) endangers to the Chinese jujube (Ziziphus jujuba Mill.) industry and it is severe.The resistant species has become to go to close the important problem.An excellent cultivar named 'XingGuang' with high resistance to JWB and sensitive cultivar 'Pozao',were used to find out two methods for isolating and identifying total RNA from young leaves and barks, and turn RNA to cDNA, the expression of resistant gene was that by the technique of cDNA-AFLP to in the research.The purpose of this study was to lay a solid foundation for further molecular biology reaserch for Chinese jujube, selecting and cloning gene fragments related to JWB and revealing the molecular mechanism of JWB resistance.This research took sensitive cultivar 'Pozao' as the comparison, studied the expression difference of the resistant variety after significant invading dyes through the field observation and the cDNA-AFLP analysis. The following results were obtained:Improved CTAB can isolate both satisfied RNA and DNA at same time from all the issues of Chinese jujube.Basing on comparative study of CTAB, PVP, β -Mercaptoethanol and Glyeogen, the optimal system for RNA isolation is:2%CTAB,4mol/Lguanidine Isomiocyanate,2.0mol/LNaCl,100mmol/LTris-HCl(pH8.0),20mmol/LEDTA(pH8.0),2o/oP VP, 2% P-Mercaptoethanoland and250ug/mlGlyeogen.The scion materials were inoculated with phytoplasma by graft. The phytoplasma was detected with PCR and fluorescence.The resistant and sensitive scion cultivars showed significant.The phytolasma can be detected both in the resistant and sensitive scion cultivars after 45d, but only in the sensitive cultivar after 96d.This period may be the key stage when the resistant gene was induced by the phytoplasma expressed. It should be the important stage in mRNA differential display analysis.The system of the reverse transcription was established for Chinese jujube, The fragments of double chains cDNA were obtained through the transpositional reaction, the length is between the 100~2500 bp which arrived the experiment request.The optimum cDNA-AFLP system was established for Chinese jujube.Pass to take the Chinese jujube cDNA as the template, The amplification of AFLP was researched ,theAFLP system is: 5 uL template cDNAs in 20p, L reaction systems ,2 p. L10 x PCR buffers, 2.5 mMdNTP1.6 uL, 5 UTaqDNA polymerase 0.15^ L, EcoRI(50 ng/ uL)0.8 uL,MttJl(50 ng/ \i L) 0.8uL and H2O9.65p, L.The procedure respond is:The amplification performed 2 min at 94 "C for the Chinese jujube cDNA thermal predenaturalizaion;50s at 94 *C for denaturalizing, 30s at 65*C for annealing(dropped 0.7*C at every cycle), 60s at 72'C for extension for 12 cycles;30s at 94*C for denaturalizing, 30s at 56*C for annealing, 30s at 72'C for extension for 23 cycles and a final extension at 72 °C for 2 min after the last cycle.Only 6 out of thirty-six pairs primers screened for cDNA-AFLP analysis amplified polymorphic bands on the resistance gene of JWB after phytoplasma inoculating,six primer pairs are E-AT/M-CCQE-AT/M-CAT,E-AG/M-CCG,E-AG/ M-CCA,E-TC/M-CAT and E-TC/M-CCT nineteen differential expression bands were gained in this research.