The Physical Locations Of REF, RT And SRPP Gene In Hevea Brasiliensis

Hevea brasiliensis has been the only commercial source of natural rubber, which is also an important strategic material and economic crop for tropical and subtropical area. More than 40 types of important genes or cDNAs about latex production and stressing resistance have been cloned, but few of them has been reported physically located onto chromosomes. Gene localization research is very important to the chromosomal physical mapping, establishing the genetic linkage groups and the corresponding relationship of the specific chromosomes, and combining genetic map with physical map. Rubber elongation factor (REF) gene was physically located onto chromosomes in Hevea brasiliensis by in situ PCR, while the rubber transferase (RT) gene and the small rubber particle protein (SRPP) gene were physically located by in situ hybridization in this study. The results were main as follows.The method on how to make chromosome speciment from the leaf of hevea brasiliensis was discussed and a complete set of method was found in this study.We made the improvement to predecessors' in situ PCR, and established an in situ PCR method suited for the gene specific sequence from hevea brasiliensis. This improved method had characteristics of low background and low false positive.The fluorescence in situ hybridization (FISH) suited for single copy and low copy gene from Hevea brasiliensis was founded. The total volume of hybridization solution was 20μl, which contained 50% deionized formamide, 10ng/μl probe,10% dextran sulfate,2×SSC, 250ng/μl sperm DNA and 0.5% SDS.The operational process was 0.1M HCl 2 min→pepsin treated 10 min→94℃bathed 1 min→70% deionized formamide 70℃denatured 2 min→ethanol dehydrated→hybridization solution denatured and ice bathed→90℃co-denatured 10 min→hybridized→signal detected.Two signals were detected on the different phases of the of Reyan 7-33-97 through the improvement in situ PCR.The REF gene was located on the long arm of chromosome 3, the percent distances from centromere to detection site was 34.51.The double-colour FISH system was found in this study, which was utilized to located the RT gene and SRPP gene on chromosomes from Reyan 7-33-97 simultaneous for the first time. The RT gene was located on the long arm of chromosome 6, the percent distances from centromere to detection site was 42.02, when the SRPP gene was located on the long arm of chromosome 8, the percent distances from centromere to detection site was 56.93.