中药地枫皮及其伪品假地枫皮的DNA条形码鉴定

目的:评价和比较几个常用DNA条形码候选序列对地枫皮及伪品假地枫皮的鉴定作用。方法:采集不同产地的地枫皮及假地枫皮样品,进行总DNA提取,选取核基因内转录间隔区2(ITS2)序列、叶绿体rbcL,mat K基因序列进行聚合酶链式反应(PCR)扩增,纯化产物测序,利用Condon Code Aligner V3. 7. 1校对拼接。结果:地枫皮及假地枫皮rbcL序列进行多次PCR扩增及测序结果均不理想,初步推测地枫皮及假地枫皮rbcL序列太长,进化较慢,不适合作为地枫皮及假地枫皮种间鉴别;地枫皮及假地枫皮的matK基因序列测序成功率分别为0和76. 8%,可能是不同类群的植物matK序列的引物标准不一;对地枫皮及假地枫皮的ITS2序列PCR扩增及测序结果最为理想,测序的成功率分别为89. 3%及91. 2%,对测序结果序列进行分析,地枫皮的ITS2序列总长度均为268个碱基,存在2个变异位点;假地枫皮的ITS2序列总长度均为430个碱基,存在4个或3个变异位点。实验结果说明地枫皮及假地枫皮ITS2序列较短,有明显的变异性,便于扩增,表明ITS2序列用于地枫皮及假地枫皮的分子鉴定优于rbcL和matK序列。结论:ITS2序列作为标准的DNA条形码能够有效地鉴定地枫皮及其伪品假地枫皮。

Identification of Illicium difengpi and Its Fake I. jiadifengpi Using DNA Barcoding

Objective:To evaluate and compare the identification of several DNA barcoding candidate sequences on Illicium difengpi and its fake I. jiadifengpi. Method:Samples from different origins of I. difengpi and I. jiadifengpi, were collect extraction of total DNA,nuclear gene ITS2 sequence,chloroplast rbcL,matK gene sequence were selected for PCR amplification,product purification and sequencing,and CondonCode Aligner V3.7.1 was used to proofread stitching. Result:PCR amplification and sequencing of rbcL sequences of I. difengpi and I. jiadifengpi were not satisfactory. It is assumed that their rbcL sequences were too long with slow evolution,which was unsuitable for interbreeding. The success rate of matK sequencing of I. difengpi and I. jiadifengpi was 0 and 76.8%,which may be because primer standards were different for matK sequences of different groups. The results of PCR amplification and sequencing of ITS2 on I. difengpi and I. jiadifengpi were successful,with the success rate of sequencing was 89.3% and 91.2%. Analysis sequencing results, the total length of ITS2 sequences was 268 bases,and there were 2 variation sites of I. difengpi. The total length of ITS2 sequences was 430 bases,and there were 4 or 3 variation sites of I. jiadifengpi. It shows that ITS2 sequences of I. difengpi and I. jiadifengpi were short and has obvious variability and can be amplify,that ITS2 sequence was better than rbcL and matK sequence in molecular identification of I. difengpi and I. jiadifengpi. Conclusion:DNA barcoding based on ITS2 sequence was a powerful and efficient tool for identification of I. difengpi and its fake I. jiadifengpi.