Isolation Of Total RNA And Establishment Of Reverse Transcription System For Chinese Jujube Fruit

Chinese jujube (Ziziphus jujuba Mill.) is important and specific fruit in China. Its fruit is rich in nutrition and loved by people. Because it can grow in many awful environments, it is ideal economic species in mountain and valley areas. In this research it was established that a method of isolation of total RNA from Ziziphus jujuba Mill. cv. wuhexiaozao and Ziziphus jujuba Mill. cv. jinsixiaozao fruit which were from different grown time. Also the Reverse Transcription System for Chinese jujube fruit was established in the research. The purpose of this research was to lay a solid foundation for cDNA-AFLP analysis, to illustrate the mechanism of stoneless of Chinese jujube, to clone the gene to stoneless, to cultivate new Chinese jujube cultivars by gene modified breeding. The result of this research was as follows:1. On the base of comparing many thesis on isolation of total RNA, a new protocol, which was entitled the improve SDS protocol, was designed in this research. By comparing the result of between modified CTAB protocol and modified SDS protocol for isolating total RNA from Chinese jujube, it was found that modified SDS protocol was better than another on the quality of total RNA. Furthermore, for improving the modified SDS protocol, it was compared that the most appropriate weight of Chinese jujube, comparison of the effect of depositing total RNA by absolute ethanol and isopropanol, the effct of wiping off polysaccharide by LiCl, the result of different sequence to deposit total RNA between absolute ethanol and isopropanol. Optimal system of isolating total RNA from Chinese jujube was established. Total RNA isolated by the system was high quality. OD₂₆₀/ OD₂₈₀ was at the range between1. 8 and 2. 0, and OD₂₆₀/ OD₂₃₀ was out of 2. 0. And isolation of total RNA from Ziziphus jujuba Mill. cv. wuhexiaozao and Ziziphus jujuba Mill. cv. jinsixiaozao used the system.2. In different developmental period, the quantity of total RNA from Chinese jujube fruit was different. And it was most large in primal period.3. It was compared the effect of depositing total RNA by absolute ethanol and isopropanol. Make sure the optimal protocol for depositing total RNA used 2.5 times volumes absolute ethanol.4. It was established that the purification system of total RNA from Chinese jujube. Make sure the optimal quantity of DNase was 10U in 40 u L system.5. System for reverse transcript cDNA was established on the study. The optimal systems were:system for the first strand cDNA synthesis : total volume 10 uL, including 5uL pured RNA, 1.625uL Oligo (dT)i5, 0.125uLRibonuclease Inhibitor (40U/uL), luL lOmmol/uL dNTPs, 2uL AMV Reverse Transcriptase 5* Reaction Buffer, 0.25uL AMV Reverse Transcriptase (lOU/uL);system for double strand cDNA synthesis: total volume 30uL, including lOuL the first strand mix, 3uL DNase 110* Reaction Buffer, 2uL DNA polymerase K4U /uL ), 1.5uL 10mmol/M.L dNTPs, 0.5uL RNase H(60U/uL), 3\iL T4 DNA Ligase 10x Reaction Buffer, 9.8uL ddH2O, 0.2pL T4 DNA Ligase.