Optimization Of CDNA-AFLP System In Ziziphus Jujuba Mill. And Its Application On Screening Of Gene Bands Related To Resistance To Jujube Witches Broom Disease

Chinese jujube (Ziziphus jujuba Mill.) is a kind of important and specific fruit tree inChina. Jujube witches'-broom disease (JWB), caused by phytoplasma, is one of the mostdestructive diseases in Chinese jujube. To utilization of cultivars with high resistance toJWB, it is important to understand resistant mechanism and screen the resistant gene byusing of molecular biology method.The diseased Ziziphus jujuba Mill. cv. pozao (a leading cultivar of Chinese jujube)was used as the standard sensitive cultivar in this study. Ziziphus jujuba Mill. cv.Xingguang with high resistance to JWB was used to screen the resistant bands related ofJWB by complementary DNA Amplified Fragments Length Polymorphism (cDNA-AFLP). The main results were as follows:1 An improved CTAB-LiCl method was established to isolate total RNA ofChinese jujube. Because Chinese jujube are rich in polysaccharides and polyphenolcompounds, the CTAB method was improved by adding polyvinylpyrrolidone(PVP) andβ-ME to remove polyphenol compounds; adding KAc and ethanol to removepolysaccharides and adding LiCl to deposit RNA. The bands of the isolated RNA wereregular and clear. The A₂₆₀/A₂₈₀ value of total RNA were between 1.8-2.0 and A₂₆₀/A₂₃₀were more than 2.0. That proved the method could extract high-purity and complete RNAat a high productivity and could remove protein, polysaccharides and polyphenolcompounds completely. The optimum system contain 2.0% CTAB, 1.4mol/L NaCl,100mmol/L Tris-Cl(pH8.0), 20mmol/L EDTA(pH8.0), 2.0% PVP-40 and 2.0%β-Mercaptoethanol (β-ME).2 An optimized cDNA-AFLP system and the PCR program suitable for Chinesejujube were established. After digested using restriction endonuclease EcoR I and Mse I,and jointed with the relevant anchor, the cDNA pool were used as primary template forpre-amplification. The pre-amplification was diluted to the concentration of 1ng/μl, whichused as the template for the selective amplification. Through groping and optimizing thereaction of AFLP selective amplification, the optimum system was established as follows:5μl template in 20μl reaction systems, 21μl 10×PCR buffer, 2μl MgCl₂(25mM), 10mMdNTP 0.4μl, 5U Taq DNA polymerase 0.12μl, EcoR I selective primer (50 ng/μl)1μl, MseI selective primer (50 ng/μl)1μl. PCR program was that the first cycle performed as 2 min at94℃to predenature the templet, then keep 94℃for 30s to denature, 65℃for 30s to anneal(dropped 0.7℃next cycle), 72℃for 1 min to extend, preceding process for 12 cycles;then 94℃for 30s to denature, 56℃for 30s to anneal, 72℃for 1min to extend for 31cycles and a final extension at 72℃for 7 min after the last cycle.3 Twenty-four differential expression bands were screened in this research. Onlyten pairs out of fifty pairs primers screened for cDNA-AFLP analysis were amplifiedpolymorphic bands on the resistance gene of JWB after phytoplasma inoculated.Twenty-four differential expression bands were screened out. Ten bands were inducedexpression and fourteen bands were restrain expression.