进口黄檀属木材DNA提取与分子鉴定方法初步研究

摘要

由于黄檀属Dalbergia种类较多,很多种类通过形态学方法难以区分,给中国进口木材检验鉴定工作带来了很大困难.为了研究黄檀属木材的分子鉴定方法,选取进口木材中常见的7种黄檀属木材,通过比较不同的木材DNA提取和纯化方法,摸索出了适合于黄檀属木材的十六烷基三甲基溴化铵(CTAB)十二烷基硫酸钠(SDS)磁珠相结合的DNA提取和纯化体系,利用巢式聚合酶链式反应(PCR)扩增出433 bp的matK基因片段,共发现12个碱基位点差异,可以将7种进口黄檀属木材及我国的降香黄檀Dalbergia odorifera逐一区分开,为黄檀属木材分子识别鉴定研究工作奠定了良好的基础.%Establishing a reliable and cost effective method to identify and distinguish wood is important in preventing the illegal timber trade.To overcome the difficulty of distinguishing Dalbergia,which consists of many valuable species,by morphological methods,molecular genetic tools to control species identity were studied.Seven species of Dalbergia wood,D.melanaoxylon,D.oliveri,D.retusa,D.louvelii,D.greveana,D.cochinchinensis,D.cultrate,imported from different countries and one domestic species D.odorifera were selected for testing.Next,an array of different wood DNA extraction and purification methods,including DNeasy Plant Mini Kit,hexadecyltrimethylammonium bromide (CTAB),sodium dodecyl sulfate (SDS) and Magnetic Beads based method,were tried; For the amplification of DNA from wood samples,two PCR reactions were successively performed (nested PCR) and allowed the amplication of one coding region of chloroplast DNA (cpDNA).Results showed that a CTAB-SDS-Magnetic Beads based method was available for Dalbergia wood DNA extraction.Then,433 bp matK gene fragments were amplified and sequenced from the seven species,of which a total of 12 single nucleotide polymorphisms were found.These polymorphic sites were able to distinguish the eight Dalbergia species.Thus,establishment of this method for DNA isolation and polymerase chain reaction (PCR) amplification makes Dalbergia wood amenable to DNA-based identification.