Cloning And Analysis Of Specific DNA Sequences Of Camellia Oleifera

Camellia oleifera is the main oil tree in those provinces which in the south of Yangtze River,there are many excellent variety has been found. In this paper, using two kinds of molecularmarker methods ISSR and SRAP for genetic analysis. Many specific DNA sequences werefound in a few breeds, recoverying, cloning and analysing this sequencing. The main results andconclusions were showed as follows:(1) The traditional methods of DNA extraction had been improved, useing1.5ml centrifugetube, drug concentration and reaction process had been improved, not only to reduce the use ofmaterials and drug but also reduced the content of impurities, quality of this method fully inaccordance with the requirements of molecular marker.(2) In this experiment, use two kinds of molecular marker method ISSR and SRAP.Ultimately reaction system were been selected through the experimental comparison. ISSR systemoptimization result was the amount of DNA template was30ng, Mg2+concentration of2.0mm/l,dNTP concentration of0.2mm/l, Taq polymerase concentration of0.5u, Primer concentration of1.5μ m/l, total reaction system was20μL, annealing temperature in the reaction process was55℃for45s; SRAP system optimization result was the amount of DNA template was30ng, Mg2+concentration of2.25mm/l, dNTP concentration of0.3mm/l, Taq polymerase concentration of1u,each Primer concentration of1.0μ m/l, total reaction system was20μL. annealing temperaturewas35℃for1min in cycles1and55℃f or1minin cycles2.(3)13specific DNA sequence was found in ISSR molecular marker, Through recoverying,cloning, sequencing and sequence analysis.the longest segquence of1509bases, the shortestsequence only174bp; Each sequence can find similar gene sequence, the highest similarity canreached100%, the similarity coefficient of the rest between70%-80%; Consensus sequence wasvery shorter, about40%of the full sequence, in sequencepaired comparison, only a few of allsequence has no gaps, most of them had gaps, with a maximum of10gap; More than one OpenReading Frames were found in most sequences, the largest can encoded a306amino acid peptide,the least also can encoded a41amino acid peptide; Comparison in NCBI, a total of8sequencescan be matched with similar amino acid peptide.(4)7specific DNA sequence was found in SRAP molecular marker, Throughrecoverying,cloning, sequencing and sequence analysis.7sequence length between300bp-610bp; Each sequence can find similar gene sequence, Similarity coefficient mainly focu on80%, thehighest similarity can reached100%; Consensus sequence was not very shorter, about400bases,have the whole series66%, all of those sequence had gaps in sequencepaired comparison, with amaximum of24gap, a minimum of1; Very sequence can found one or more Open ReadingFrames, the largest can encoded a83amino acid peptide, the least also can encoded a27aminoacid peptide; Comparison in NCBI, a total of4sequences can be matched with similar amino acidpeptide.(5) Specific sequences that can be used as an effective method for the identification ofGermplasm Resources.