Isolation Of Total RNA And Establishment Of DDRT-PCR System For ZiZiphus Jujuba Mill

Chinese jujube(Ziziphus jufuba Mill) is important and specific fruit tree in china. An excellent cultivar named 'Kangfeng NO.1' with high resistance to "jujube witches' broom"(JWB) disease and sensitive cultivar 'Pozao', were used to find out proper methods for isolating and identifying total RNA from plantlets, young leaves, barks, root skins and fruits , the suitable conditions for storing RNA. Furthermore, DDRT-PCR technique system for Chinese jujube was firstly established .The purpose of this study was to lay a solid foundation for carrying out further molecular biology reaserch for Chinese jujube, selecting and cloning gene fragments related to JWB and revealing the molecular mechanism of JWB resistance.The methods of isolating total RNA from plant tissues, including Improved CTAB, SDS-phenol, Hot borate, Guanidine Isothiocyanate and Trizol kit method, were compared and improved. According to quality, yield, time and cost etc. Trizol kit is the optimal method to isolate RNA from tissue-culture plantlets, by which high-quality total RNA of OD₂₆₀/OD₂₈₀=1.82 and OD₂₆₀/OD₂₃₀=2.02, can be isolated within 1 hour, and the yield can reach 55μg/g. Both Improved CTAB and SDS-phenol can isolate high quality RNA from field leaves, of which Improved CTAB can get better quality and SDS-phenol can obtain higher yield. All of the five methods are suitable to isolate RNA from barks, Improved CTAB and SDS-phenol showed best in RNA quality and Trizol kit was best in RNA yield; Improved CTAB and SDS-phenol can obtain reasonable RNA from root skins whose RNA is less and difficult to extract, and SDS-phenol method is better in view of RNA yield; The satisfied RNA of fruit can be obtained only by Improved CTAB method(OD₂₆₀/OD₂₈₀=1.82 , OD₂₆₀/OD₂₃₀=2.92, the yield less than 17.0μg/g). 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 are: 2%CTAB, 4mol/LGuanidine Isothiocyanate, 2.0mol/LNaCl, 100mmol/L Tris-HCl(pH8.0), 20mmol/L EDTA(pH8.0), 2%PVP, 4% P-Mercaptoethanol and 250ug/mlGlyeogen.The tissues of Chinese jujube used for RNA isolation have different storge life under different condition: 4℃:,10d; -20℃,60d; -70℃, more than 90d. The purified RNA can keep quality for nearly 60d in TE buffer or DEPC-H₂O, approximately 90d in 0.5% SDS buffer, and more than 6 months in Formamide without ions under -20°C; and nearly 7 months in DEPC-H₂O or TE buffer, more than 12 months in 0.5% SDS buffer and...