Establishment Of RT-PCR System For Apple Cultured In Vitro And Study On Expression Of Exogenous CpTI Gene On MRNA

Using translated CpTI gene apple(Malus domestica Borkh) in vitro (including Fuji, Jonagold, Orin and Royal Gala) as experiment materials, the techniques of detecting translated CpTI gene from apple cultured in vitro by RT-PCR were studied. Firstly, extracting total RNA from grapevine tisste.Secondly, the cDNA of CpTI gene were synthesized with reverse transcriptase using 3'primer, and were amplified by PCR using two primers. Thirdly, the. fragments of PCR products were sequenced and analysised. In this search , a series of methods of extracting total RNA in plants were tried, compared and modified. And the systerm of detecting foreign gene was set up. The results are as following:1. The quality of RNA is the base the process of RT-PCR, we need pure and undegradated RNA. Phenolic compounds, polysacchrides, proteins and secondary metabolites are the main factors interfering the isolation of total RNA from apple cultured in vitro. In this study , five methods(RNAplant kit, CTAB-guanidinium , modified CTAB, and Hot borate methods) were compared and modified, According to quality, yield, time and cost etc, modified CTAB method is the optimal method to isolate total RNA from apple cultured in vitro, and its extracting buffer includes 2%CTAB, 2.0 mol/L NaCl, 100 mmol/L Tris-HCl (pH 8.0), 20mmol/L EDTA (pH 8.0), 2%β-Me(adding before using). This method can eliminate Phenolic compounds, polysacchrides, proteins, and obtained pure, high quality RNA which is fit for RT-PCR within 3 hours, and made the detection successful.2. After exorcizing DNA from RNA by DNase I, A suitable reverse transcription system for apple cultured in vitro was established, through which the synthesized cDNA fragment was 100bp to 1500bp, and can be satisfied for the requirement of PCR of cDNA.3. The 5'and 3'primers of CpTI gene were synthesized according to the published sequence. The reverse transcription step was performed with total RNA extracted from apple cultured in vitro. From the synthesized first cDNA strand, 309 bp nucleotide fragments were amplified by two primers, which are the same long as what we had expected.4. An efficient RT-PCR system for apple cultured in vitro was established. The system is as follows: in RT systerm, the primer consentration is 0.8 umol/L, and the total RNA consentration is 1.0 ug. Total volume of PCR is 25 uL, containing 0.6 umol/L up-stream primer and down-stream primer, 300 umol/L dNTPs, 1.6 mmol/L Mg2+, 1.5 U Taq enzyme. The program of PCR is 94°C, 2mins;94°C, 50s, 55 °C, 50s, 72 °C, 100s;30cycles;72 °C,10mins;conservation at 4°C.5. Testing the expression of exogenous CpTI by RT-PCR method in 60 apple cultured in vitro samples, and the results are as follows: There are not wanted bands in 54 and 60 samples, the wanted bands in 42-50 ^ 52 and 55~59 samples are dim, and the wanted band in other 43 samples are bright.