Studies On Eriobotrya Japonica ISSR Hereditary Difference Analysis And Chemical Constituents Content Correlation

OBJECTIVE:To study the ISSR-PCR identify fingerprint and quality correlation of Eriobotrya japonica from different strains.METHODS:1. Collect Fujian Putian in 24 different strains loquat leaf.2. Using an improved CTAB method extraction separation loquat leaves total genomic DNA, to test DNA concentration and purity with 0.8% agarose gel electrophoresis and uv spectrophotometer.3. Screening ISSR-PCR the template concentration, primer concentration, dNTP concentration and Taq enzyme dosage, optimizing ISSR-PCR system.4. For primer screening, first elected can produce clear strip primers, and then from these primer extract can have polymorphism primers, and these produced by PCR polymorphism of statistics, then the bands of the genetic distance between different samples were analyzed.5. Through the distance coefficient by UPGMA cluster analysis method to analyze electrophoresis to construct assorts product relationship tree.6. Adopt HPLC detected 24 different strains of Eriobotrya japonica leaf medicinal materials of oleanolic acid and ursolic acid content, to study the ISSR-PCR identify fingerprint and quality correlation.RESULTS:1.24 strains loquat leaf medicinal materials classics appraisal are rosaceae Eriobotrya japonica (Eriobotrya japonica leaves (Thunb.) Lindl.) leaves.2.Improved CTAB method extraction and separation loquat leaves after total genomic DNA electrophoresis mobility presents a small neat bands, without the diffuse fluorescence are appears, the extraction of DNA samples than pure, basic no degradation and RNA pollution, suited to ISSR amplification.3. Through investigation for template DNA concentration, DNA polymerases dosage, primer concentration, dNTP concentration of inspection, the optimized PCR reaction system:total response volume 25ul, including 60ng template DNA, DNA polymerases dosage for 0.9 U, primer eventually concentration of 0.4 mol/l, speeds dNTP eventually concentration for 200μmol/l,10 x Buffer for 2.5μl, join sterilization deionized water to 25μl.4. From 100 the ISSR primers filtrated polymorphisms good 14 primers, analyzing different primer amplification of the bands in 24the different strains of loquat leaves were identified.5. Using the genetic distance, the clustering method is tested 24 different strains of loquat leaf is divided into two big branch. Use primer S859, S856, S818, S822, the method of combining the different strains of 24 were identified.6.The content determination results indicate:different strains of loquat leaves have much different contents of Oleanolic acid and Ursolic acid by HPLC.And Oleanolic acid and Ursolic acid content of the lowest and highest strains is consistent. Oleanolic acid and Ursolic acid content of the lowest 8 loquat leaves,in recent between 400bp and 500bp,missing with a specific band,but the number 14 and 13 have no this band,so we preliminary think it related to the quality of loquat leaves. ISSR PCR fingerprint reveals the loquat have close kin, contains leaf strains of oleanolic acid and ursolic acid content also relatively similar, hint this has certain correlation.CONCLUSION:1.Different strains of loquat leaf medicinal materials exist the genetic differentiation, ISSR markers can be used as a different strain loquat leaves identify molecular markers.2.The different strains loquat leaves of oleanolic acid and ursolic acid content and genetic relationship exist certain correlation.3.ISSR-PCR can at the molecular level for loquat leaves provide scientific basis for quality evaluation.