Please wait a minute...
浙江大学学报(医学版)  2018, Vol. 47 Issue (3): 300-306    DOI: 10.3785/j.issn.1008-9292.2018.06.14
中药现代化     
松针的显微及分子鉴别研究
龚恒佩1(),刘祖望1,陈妍月1,张坚2,程汝滨1,黄真1,*()
1. 浙江中医药大学药学院, 浙江 杭州 310053
2. 天津中医药大学药学院, 天津 300193
Microscopic and molecular identification of pine needles
GONG Hengpei1(),LIU Zuwang1,CHEN Yanyue1,ZHANG Jian2,CHENG Rubin1,HUANG Zhen1,*()
1. College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
2. College of Pharmaceutical Sciences, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
 全文: PDF(5049 KB)   HTML( 4 )
摘要:

目的: 利用显微和分子鉴定技术对不同植物基源的松针药材进行鉴别,为松针的鉴别提供科学依据。方法: 采用基源、显微鉴定的方法研究马尾松(Pinus massoniana Lamb.)、黑松(Pinus thunbergii Parl.)和华山松(Pinus armandii Franch.)的特征;利用PCR扩增和双向测序分析植物DNA条形码ITS2rbcL序列特征;采用分子进化遗传分析(MEGA)6.0软件进行种间、种内Kimura2-parameter(K2P)遗传距离计算,并构建邻接系统聚类树。结果: 三种松针数目及长度、维管束数、气孔线的分布、树脂道数目及分布等具有明显区别。马尾松、黑松和华山松的ITS2序列长度分别为470、469和470 bp,rbcL序列长度均为553 bp;ITS2序列在马尾松、黑松和华山松的种内变异率分别为0%、0.2%和2.8%,rbcL序列在这三种松针样品的种内变异率分别为0%、2.4%和1.1%。ITS2序列在松针的种内和种间遗传距离中不存在显著的条形码间隔;而rbcL序列的种内和种间距离存在一定的条形码间隔。邻接系统树结果表明,三种松针药材聚为不同的分支,rbcL条形码序列可鉴别马尾松、黑松、华山松及其近缘的植物。结论: 利用基源、显微及分子鉴定技术可准确、快速地鉴别马尾松、黑松和华山松,为松针的质量评价和分类鉴别提供了方法和依据。

关键词: 松科/化学序列分析药材显微鉴定自动数据处理    
Abstract:

Objective: To identify pine needles from different plant origins by microscopic and molecular approaches. Methods: The characteristics of pine needles of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were investigated via plant morphology and microscopic characteristics. ITS2 and rbcL were analyzed with PCR amplification and bi-directional sequencing. MEGA 6.0 was used to calculate the intra-and inter-specific Kimura-2-Parameter (K2P) distances, and the phylogenetic tree was constructed by using the neighbor-joining (NJ) method. Results: There were significant differences in the number and length of pine needles, number of vascular bundles, distribution of stomatal lines, number and distribution of resin channels among three kinds of pine needles. The lengths of ITS2 sequences of Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 470, 469 and 470 bp, respectively. The lengths of rbcL sequences in three kinds of pine needles were 553 bp. The intraspecific variation rates of ITS2 sequences in Pinus massoniana Lamb., Pinus thunbergii Parl. and Pinus armandii Franch. were 0%, 0.2%, and 2.8%, respectively; and the intraspecific variation rates of rbcL sequences were 0%, 2.4%, and 1.1%, respectively. There was no significant barcoding gap in intraspecific and interspecific genetic distances of ITS2 sequences. The intraspecific and interspecific distances of rbcL sequences were clearly separated in the barcoding gap test. The NJ tree based on rbcL showed that the three pine needles clustered into three separate groups, indicating that rbcL DNA marker could distinguish the Pinus massoniana Lamb., Pinus thunbergii Parl., Pinus armandii Franch. and its close relative species. Conclusions: The three types of pine needles can be distinguished accurately and rapidly by microscopic and molecular identification. The study provides methodology and experimental basis for the quality evaluation and classification of pine needles.

Key words: Pinaceae/chemistry    Sequence analysis    Microscopical identification of crude drug    Automatic data processing
收稿日期: 2018-02-12 出版日期: 2018-09-18
CLC:  R282.5  
基金资助: 国家自然科学基金(81573643)
通讯作者: 黄真     E-mail: gonghengpei1994@163.com;huangzhen@zcmu.edu.cn
作者简介: 龚恒佩(1994-), 女, 硕士研究生, 主要从事中药品质评价及资源开发利用研究; E-mail:gonghengpei1994@163.com; https://orcid.org/0000-0001-9319-2985
服务  
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章  
龚恒佩
刘祖望
陈妍月
张坚
程汝滨
黄真

引用本文:

龚恒佩,刘祖望,陈妍月,张坚,程汝滨,黄真. 松针的显微及分子鉴别研究[J]. 浙江大学学报(医学版), 2018, 47(3): 300-306.

GONG Hengpei,LIU Zuwang,CHEN Yanyue,ZHANG Jian,CHENG Rubin,HUANG Zhen. Microscopic and molecular identification of pine needles. J Zhejiang Univ (Med Sci), 2018, 47(3): 300-306.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.06.14        http://www.zjujournals.com/med/CN/Y2018/V47/I3/300

图 1  三种松针的形态图
松针来源 松针长度(cm) 松针类型 松针形态 叶鞘
马尾松 12~20 两针或三针一束 腹面凹下成槽,背面拱圆,叶缘有细齿 褐色、宿存、质地柔软
黑松 6~12 两针一束 腹面平整,背面拱圆,叶缘有细齿 褐色、宿存、质地粗硬
华山松 8~15 五针一束 边缘有细齿 脱落、质地细柔
表 1  三种松针的形态和质地比较
图 2  三种松针的横切面及气孔显微图像
松针来源 横切面形状 维管束数 维管束类型 树脂道数 树脂道类型 凹陷气孔数
马尾松 半圆,腹面弧形稍内陷 2 外韧型;“八”字型排列 8 边生 腹面分别为3、4个;背面5~7个
黑松 半圆,腹面弧线向外 2 外韧型;“八”字型排列 6 中生 腹面6~10个;背面8~15个
华山松 三角形,腹面角度小于直角 1 外韧型 3 中生 两腹面气孔数各为4个;背面无气孔
表 2  三种松针显微镜下特征比较
松针来源 序列类型 长度(bp) 平均GC含量(%) 保守位点(个) 变异位点(个) 信息位点(个) 转换(个) 颠倒(个) 变异率(%)
GC含量:鸟嘌呤和胞嘧啶所占的比例.
马尾松 ITS2 470 58.58 469 1 0 0 0 0.2
rbcL 553 43.76 553 0 0 0 0 0
黑松 ITS2 470 58.51 470 0 0 0 0 0
rbcL 553 43.58 553 0 0 0 0 0
华山松 ITS2 469 57.25 468 1 0 1 0 0.2
rbcL 553 44.42 553 1 1 1 0 0.2
表 3  三种松针ITS2和rbcL序列特征比较
图 3  基于 ITS2 、rbcL序列构建的松针邻接系统树
1 全国中草药汇编写组 . 全国中草药汇编:上册[M]. 北京: 人民卫生出版社, 1976: 495
National Chinese Herbal Writing Group . National compilation of Chinese herbal medicine:volume one[M]. Beijing: People's Medical Publishing House, 1976: 495
2 江苏新医学院 . 中药大辞典:上册[M]. 上海: 上海科学技术出版社, 1986: 1254
Jiangsu New Medical College . Chinese materia medica dictionary:volume one[M]. Shanghai: Shanghai Scientific & Technical Publishers, 1986: 1254
3 李时珍 . 本草纲目[M]. 北京: 人民卫生出版社, 2004: 1917
LI Shizhen . Compendium of materia medica[M]. Beijing: People's Medical Publishing House, 2004: 1917
4 ROGACHEV A D , SALAKHUTDINOV N F . Chemical composition of Pinus sibirica (Pinaceae)[J]. Chem Biodivers, 2015, 12 (1): 1- 53
doi: 10.1002/cbdv.v12.1
5 ZHAN R , WU J , Ouyang J . In vitro antioxidant activities of sodium zinc and sodium iron chlorophyllins from pine needles[J]. Food Technol Biotechnol, 2014, 52 (4): 505- 510
doi: 10.17113/ftb
6 BASHOLLI-SALIHU M , SCHUSTER R , HAJDARI A et al. Phytochemical composition, anti-inflammatory activity and cytotoxic effects of essential oils from three Pinus spp[J]. Pharm Biol, 2017, 55 (1): 1553- 1560
doi: 10.1080/13880209.2017.1309555
7 国家药典委员会 . 中华人民共和国药典(2015年一部)[M]. 北京: 中国医药科技出版社, 2015: 206
National Pharmacopoeia Commission . People's Republic of China pharmacopoeia(2015 volume Ⅰ)[M]. Beijing: China Medical Science Press, 2015: 206
8 聂奇华, 张志琴 . 云南松松针生药学鉴别研究[J]. 海峡药学, 2017, 29 (6): 21- 22
NIE Qihua , ZHANG Zhiqin . The pharmacognostic studies on Pinus Yunnanensis needles[J]. Strait Pharmaceutical Journal, 2017, 29 (6): 21- 22
doi: 10.3969/j.issn.1006-3765.2017.06.009
9 郭春晓.油松松针的化学成分和组织形态学研究[D].沈阳: 沈阳药科大学, 2009.
GUO Chunxiao. Studies on chemical constituents and histomorphology of Pinus tabulaeformis Carr. needle[D]. Shenyang: Shenyang Pharmaceutical University, 2009. (in Chinese)
10 黄璐琦, 袁媛, 袁庆军 et al. 中药分子鉴定发展中的若干问题探讨[J]. 中国中药杂志, 2014, 39 (19): 3663- 3667
HUANG Luqi , YUAN Yuan , YUAN Qingjun et al. Discussion on some problems in the development of molecular identification of Chinese traditional medicine[J]. China Journal of Chinese Materia Medica, 2014, 39 (19): 3663- 3667
11 CHEN S , YAO H , HAN J et al. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J]. PLoS One, 2010, 5 (1): e8613
doi: 10.1371/journal.pone.0008613
12 PANG X , SONG J , ZH UY et al. Applying plant DNA barcodes for Rosaceae species identification[J]. Cladistics, 2011, 27 (2): 165- 170
doi: 10.1111/cla.2011.27.issue-2
13 朱英杰, 陈士林, 姚辉 et al. 重楼属药用植物DNA条形码鉴定研究[J]. 药学学报, 2010, 45 (3): 376- 382
ZHU Yingjie , CHEN Shilin , YAO Hui et al. DNA barcoding the medicinal plants of the genus Paris[J]. Acta Pharmaceutica Sinica, 2010, 45 (3): 376- 382
14 夏至, 冯翠元, 高致明 et al. 黄芩及其同属近缘种的DNA条形码鉴定研究[J]. 中草药, 2014, 45 (1): 107- 112
XIA Zhi , FENG Cuiyuan , GAO Zhiming et al. Authentication of DNA barcoding of Scutellaria baicalensis and its related species[J]. Chinese Traditional and Herbal Drugs, 2014, 45 (1): 107- 112
15 刘美子, 刘萍, 李美妮 et al. 党参及其易混伪品的ITS2分子鉴定[J]. 世界科学技术-中医药现代化, 2011, 13 (2): 412- 417
LIU Meizi , LIU Ping , LI Meini et al. ITS2 molecular identification of codonopsis pilosula and its adulterants[J]. Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology, 2011, 13 (2): 412- 417
doi: 10.3969/j.issn.1674-3849.2011.02.037
16 杨慧洁, 杨世海, 张淑丽 et al. 药用植物DNA条形码研究进展[J]. 中草药, 2014, 45 (18): 2581- 2587
YANG HuiJie , YANG Shihai , ZHANG Shuli et al. Research progress on DNA barcodes of medicinal plants[J]. Chinese Traditional and Herbal Drugs, 2014, 45 (18): 2581- 2587
doi: 10.7501/j.issn.0253-2670.2014.18.001
17 NEWMASTER S G , FAZEKAS A J , RAGUPATHY S . DNA barcoding in land plants:evaluation of rbcL in a multigene tiered approach[J]. Can J Bot, 2006, 84 (3): 335- 341
doi: 10.1139/b06-047
18 ZHANG Z Y , YANG J B , Li D Z . Phylogenetic relationship of an extremely endangered species, Pinus squamata (Pinaceae) inferred from four sequences of the chloroplast genome and ITS of the nuclear ribosomal DNA[J]. J Integr Plant Biol, 2003, 45 (5): 530- 535
19 胡雅琴.基于rbcLmatK基因序列探讨松科系统进化的可能性及rbcLmatK基因SSCP分析[D].西安: 陕西师范大学, 2004.
HU Yaqin. The possibility of studying pinaceae phylogeny based on rbcL and matK sequences and the analysis of PCR-rbcL and PCR-matK[D]. Xi'an: Shaanxi Normal University, 2004. (in Chinese)
20 杨辰.松属五针松组物种的分子鉴定研究[D].兰州: 兰州大学, 2014.
YANG Chen. Molecular identification of species from Pinus Sect. strobus[D]. Lanzhou: Lanzhou University, 2014. (in Chinese)
21 王柯, 陈科力, 刘震 et al. 锦葵科植物DNA条形码通用序列的筛选[J]. 植物学报, 2011, 46 (3): 276- 284
WANG Ke , CHEN Keli , LIU Zhen et al. Screening of universal DNA barcodes for malvaceae plants[J]. Chinese Bulletin of Botany, 2011, 46 (3): 276- 284
22 周会, 荆胜利, 李刚 et al. 叶绿体基因组分析在植物系统发育中的应用[J]. 植物学研究, 2014, 3 (1): 1- 9
ZHOU Hui , JING Shengli , LI Gang et al. The Applications of chloroplast genome analysis in plant system development[J]. Botanical Research, 2014, 3 (1): 1- 9
[1] 楼昭涵 等. 义乌市消化道恶性肿瘤空间格局分析[J]. 浙江大学学报(医学版), 2017, 46(5): 537-545.
[2] 叶臻华 等. 宁波市鄞州区大气可吸入颗粒物暴露与冠心病就诊人数的时间序列分析[J]. 浙江大学学报(医学版), 2016, 45(6): 607-613.
[3] 季加孚. 精准医学时代肿瘤样本库的规范化建设[J]. 浙江大学学报(医学版), 2016, 45(4): 331-334.
[4] 宋炜 等. 艾滋病合并分枝杆菌感染患者分枝杆菌菌种鉴定[J]. 浙江大学学报(医学版), 2016, 45(3): 243-248.
[5] 毛小荣, 张立婷, 蒋妮, 肖萍, 彭雪彬, 张有成. 丙型肝炎病毒基因型在中国大陆汉族慢性丙型肝炎患者中的分布特征[J]. 浙江大学学报(医学版), 2015, 44(4): 417-422.
[6] 郝爱平. 人类pygo1基因的生物信息学分析[J]. 浙江大学学报(医学版), 2014, 43(4): 453-457.
[7] 楼敏. 缺血性卒中患者的溶栓治疗:问题和对策[J]. 浙江大学学报(医学版), 2014, 43(1): 1-6.
[8] 李小余, 王银环, 严杰, 程东庆. 钩端螺旋体groEL基因原核表达及其产物免疫保护作用[J]. 浙江大学学报(医学版), 2013, 42(2): 164-170.
[9] . 数码摄影在口腔科比色中的运用[J]. 浙江大学学报(医学版), 2011, 40(4): 432-435.
[10] 刘晓艳;方红;陈鸿超;蒋筱凌. CMV启动子调控的VEGF-shRNA表达载体的构建及有效干扰序列的筛选[J]. 浙江大学学报(医学版), 2010, 39(2): 181-186.
[11] 邱洁;张敏;周晓玉;程锐;曹兴国;王玢;郭锡熔. 人类肥胖相关新基因LYRM1真核表达载体的构建及稳定转染3T3-L1细胞系的建立[J]. 浙江大学学报(医学版), 2009, 38(5): 493-497.
[12] 严智宇;丁宗辉. 全基因组预测Phenylobacterium zucineum HLK1T的外排蛋白[J]. 浙江大学学报(医学版), 2009, 38(2): 174-180.
[13] 张磊;薛峰;严杰;毛亚飞;李立伟. 不同血清群问号钩端螺旋体mce基因序列分析及表达模式的研究[J]. 浙江大学学报(医学版), 2008, 37(6): 564-571.
[14] 曹清毅;侯晓丽;吴昊;陈智 . 应用系统发育分析方法鉴别近缘病原菌[J]. 浙江大学学报(医学版), 2007, 36(6): 531-536.
[15] 宁铂涛;汤永民;曹江;沈红强;钱柏芹. 抗人CD14新克隆ZCH-7-2F9单抗轻重链可变区基因克隆与序列分析[J]. 浙江大学学报(医学版), 2007, 36(4): 348-354.