Cloning Of A Retrotransposon Gene Fragment And Establishment Of The Inter-retrotransposon Amplified Polymorphism(IRAP) Molecular Marker System Of Eggplant

Plant retrotransposons have characteristics of ubiquitous distribution, high copy number and single insertion sites. It is suitable to study genome constitution, charge of expression, evolution of genome, phylogeny and biodiversity. In this study, the cloned gene fragment of eggplant(Solanum melongena L.) retrotransposon will provide appliance condition for eggplant in studying genome and retrotransposon-based molecular marker. The optimum reaction system and amplification procedure of IRAP(inter-retrotransposon amplified polymorphism)-PCR in eggplants were studied in order to analysis parent relation and gene mapping.The main results are as follows:1. PCR primers were designed depend on the reported part sequences of Solanum demissum gag-pol polyprotein and other reported structure characters of plant retrotransposon.about 518 bp DNA fragment was cloned from genome of xi′an green eggplant. Due to the results of searching the GenBank by Blast methods showed that the cloned sequence and Solanum demissum putative gag-pol polyprotein were 43% homologous in region from 90aa to 243aa,the cloned fragment would had correlation with coding of eggplant gag-pol polyprotein.2. The optimum reaction system of IRAP in eggplant was studied in order to ensure stability and reproducibility of IRAP. After testing some important influencing factors of IRAP in eggplant, the results showed that in 20μL reaction system the optimal reaction system of IRAP-PCR was as follows: 10×Buffer(100mmol/L Tris-HCl,100mmol/L KCl, 80mmol/L (NH4)2SO4 ,pH9.0,NP-40)2μl,2 mmol/L MgCl2 , 0.2 mmol/L dNTPs, Taq DNA polymerase 1.0 U, primer 0.4μmol/L,template DNA 50ng.The optimal amplification procedure was as follow: pre-denature at 94℃for 4 min,amplification was carried out in 32 cycles at 94℃for 30 sec,45℃for 30 sec and 72℃for 2 min,with final extension at 72℃for 10 min.