畲药十二时辰及其混淆品基原植物基因组DNA提取及鉴别

何舒澜; 李泳宁; 朱扶蓉

doi:10.19303/j.issn.1008-0384.2021.03.003

畲药十二时辰及其混淆品基原植物基因组DNA提取及鉴别

doi: 10.19303/j.issn.1008-0384.2021.03.003

福建卫生职业技术学院，福建　福州　350101

基金项目: 福建省卫生计生中青年骨干人才项目（2016-ZQN-75）；福建省中青年教师教育科研项目职业院校专项（JZ180548）；福建卫生职业技术学院科技创新团队项目（2018-1-3）

详细信息

作者简介:
何舒澜（1980−），男，硕士，副教授，研究方向：中草药栽培与鉴定（E-mail：181329113@qq.com）

通讯作者:
朱扶蓉（1971−），女，硕士，教授，研究方向：中药材鉴定与中药质量标准（E-mail：674747600@qq.com）

中图分类号: R 282
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出版历程
- 收稿日期: 2020-07-25
- 修回日期: 2021-02-03
- 网络出版日期: 2021-03-27
- 刊出日期: 2021-03-31

Genomic DNA Extraction for Authentication of Clematis florida Thunb. var. plena D. Don

Fujian Health College, Fuzhou, Fujian 　350101, China

摘要

摘要: 目的建立一种快速经济的提取畲药十二时辰基原植物基因组DNA的方法，实现通过分子生药学方法准确鉴别该中草药的真伪，为进一步开发应用该福建地区特色中草药奠定基础。方法采用CTAB法、改良CTAB法、试剂盒法、SDS法、高盐低pH法提取十二时辰基原植物叶片基因组DNA，采用琼脂糖凝胶电泳检测DNA的质量和完整性，用优选后的方法提取其基因组DNA，与其混淆品DNA分别进行双向测序，评价该分子生药学方法的鉴别效果。结果改良CTAB法提取得到的DNA纯度较高，SSR引物扩增与ITS2序列扩增均可得到清晰明亮的条带，是提取畲药十二时辰基原植物基因组DNA的最佳方法。用该方法提取得到的基因组DNA经PCR扩增后的产物可用于双向测序，进而鉴别它的基源，实现与常见混淆品的准确区分。结论本研究建立的改良CTAB法能有效地提取畲药十二时辰的基因组DNA，方法操作简单、提取得到的DNA质量较好，可用于畲药十二时辰与其混淆品的分子生药学鉴别。
- 十二时辰 /
- 基因组 /
- DNA提取 /
- 鉴别
Abstract: Objective A rapid and economical method to extract the genomic DNA from Clematis florida Thunb.var. plena D. Don for commercial authentication of the valuable Chinese herbal medicine was established. Method CTAB, Testing Kit, SDS, and the high salt/low pH methods, along with a modified CTAB method, were applied to extract genomic DNA from leaves of the herbal plant. The quality and integrity of the extracted DNA samples were compared using agarose gel electrophoresis. Then, by bidirectional sequencing, the selected molecular pharmacognosy method was used to differentiate between the authentic material and a counterfeit for methodology verification. Result The modified CTAB method yielded high purity DNA with clear and bright bands on SSR primer and ITS2 sequence amplifications. The PCR amplified products of the extracted DNA were used in the bidirectional sequencing to correctly identify and accurately differentiate the true and faux products. Conclusion The improved CTAB method established in this study could effectively extract the genomic DNA of the target plant material. It was simple, rapid and reliable in extracting high quality DNA for molecular pharmacognosy identification on Clematis florida Thunb. var. plena D. Don.
- Clematis florida Thunb. var. plena D. Don /
- genome /
- DNA extraction /
- identification

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图 1 DNA琼脂糖凝胶电泳结果

注：M为DNA Marker；1～2：CTAB法；3～4：改良CTAB法；5～6：SDS法；7～8：高盐低pH法；9～10：试剂盒法。

Figure 1. Agarose gel electrophoresis of DNA

Note: M: DNA marker; 1-2: CTAB method; 3-4: Improved CTAB method; 5-6: SDS method; 7-8: High salt/low pH method; 9-10: Testing Kit method. Each method was executed in duplication.

下载: 全尺寸图片幻灯片

图 2 11种引物扩增后琼脂糖凝胶电泳结果

注：1～2：引物A；3～4：引物B；5～6：引物C；7～8：引物D；9～10：引物E；11～12：引物F；13～14：引物G；15～16：引物H；17～18：引物I；19～20：引物J；21～22：引物K。

Figure 2. Agarose gel electrophoresis of 11 primers after amplification

Note: 1-2: Primer A; 3-4: Primer B; 5-6: Primer C; 7-8: Primer D; 9-10: Primer E; 11-12: Primer F; 13-14: Primer G; 15-16: Primer H; 17-18: Primer I; 19-20: Primer J; 21-22: Primer K.

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图 3 SSR扩增产物琼脂糖凝胶电泳结果

注：M：DNA Marker；2～4：试剂盒法；5～7：CTAB法；8～10：改良CTAB法；11～13：高盐低pH法；1、14：阴性对照。

Figure 3. Agarose gel electrophoresis of SSR amplification products

Note: M: DNA marker; 2-4: Testing Kit method; 5-7: CTAB method; 8-10: Improved CTAB method; 11-13: High salt/low pH method; 1,14: Negative control.

下载: 全尺寸图片幻灯片

图 4 ITS2扩增产物琼脂糖凝胶电泳结果

注：M：DNA Marker；2～4：高盐低pH法；5～7：改良CTAB法；8～10：CTAB法；11～13：试剂盒法；1、14：阴性对照。

Figure 4. Agarose gel electrophoresis of ITS2 amplification products

Note: M: DNA marker; 2-4: High salt/low pH method; 5-7: Improved CTAB method; 8-10: CTAB method; 11-13: Testing Kit method; 1,14: Negative control.

下载: 全尺寸图片幻灯片

图 5 样品6与样品7的ITS2扩增产物琼脂糖凝胶电泳结果

注：M：DNA Marker；1：样品6；2：样品7。

Figure 5. Agarose gel electrophoresis on ITS2 products of Sample 6 and 7

Note: M: DNA marker; 1: Sample 6; 2: Sample 7.

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图 6 16SrDNA基因序列系统发育进化树

A B
注：A为畲药十二时辰基原植物进化树，B为畲药十二时辰混淆品基原植物进化树。

Figure 6. Phylogenetic tree of 16SrDNA gene sequence

Note: A and B are the phylogenetic trees of Clematis florida Thunb.var.plena D.Don and its confused.

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表 1 引物信息

Table 1. Information on primer

引物编号 Primer number	引物序列（5′ to 3′） Primer sequence（5' to 3' ）	退火温度 TM/℃	产物长度/bp Product length/bp
A	F: AAATGCCAATCCAACA R: CCCGGATAAGATAAATGA	48	218
B	F: ATGTGCCATTCAAGAT R: TGGCGGGAATAGAAAT	49	184
C	F: GAAATTCCCTTGGTTG R: CATTGAAACGCTTACTT	48	269
D	F: CCGTTGTTTCTTATTT R: TGAAAGTTTCGCATTA	44	267
E	F: AAGGGATATGGATAAATGG R: TGACTAGGCCCGAACT	53	277
F	F: TGACCCAAGAAATCCC R: CCATAGGCTCATAAAGAAT	51	181
G	F: ACCGTTGGTAGAGTTT R: AATTAAGAACCGAAGC	48	284
H	F: TTGATGGGTGTTGAGG R: ACTCCTGGCTTATTTG	50	274
I	F: TTTGCGGTACTAATCT R: TATCACAAGCCCAAGT	48	246
J	F: GTAGAGGCATATTGGG R: ATGGAACGAAAGGAAA	49	295
K	F: AATGACTAGGCCCGAACT R: CAAAGGGATATGGATAAATG	53	281

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表 2 DNA的OD_260/280与模板浓度

Table 2. OD_260/280 and template concentration of DNA

提取方法 Extraction method	比值（A260/A280） Specific value （A260/A280）	质量浓度 Mass concentration/ （ng·μL⁻¹）
CTAB法 CTAB method	1.98	68.35
SDS法 SDS method	1.24	26.15
高盐低pH法 High salt and low pH method	1.68	36.10
改良CTAB法 Modified CTAB method	1.87	60.40
试剂盒法 Kit method	1.83	68.25

下载: 导出CSV

参考文献(23)

[1]	钟隐芳. 福安畲医畲药[M]. 福州: 海风出版社, 2010: 109−110.
[2]	黄泽豪, 张月玲. 畲药十二时辰的本草考证 [J]. 中国民族民间医药, 2014, 23(1):2−3, 5. HUANG Z H, ZHANG Y L. Herbal textual research on She medicine at “ twelve hours” [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2014, 23(1): 2−3, 5.(in Chinese)
[3]	朱扶蓉, 滕春福, 李清禄. 响应面法优化畲药十二时辰总皂苷超声提取工艺的研究 [J]. 时珍国医国药, 2016, 27(12):2874−2878. ZHU F R, TENG C F, LI Q L. Response surface methodology was used to optimize the ultrasonic extraction process of total saponins in She medicine at“twelve hours” [J]. Lishizhen Medicine and Materia Medica Research, 2016, 27(12): 2874−2878.(in Chinese)
[4]	张继州, 周春权, 李细彬, 等. 畲药 “十二时辰” 水煎剂的急性毒性实验研究 [J]. 中国民族民间医药, 2017, 26(8):27−28, 34. ZHANG J Z, ZHOU C Q, LI X B, et al. Experimental study on acute toxicity of She medicine “twelve hours” decoction [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2017, 26(8): 27−28, 34.(in Chinese)
[5]	周春权, 赵立, 蒋畅, 等. 畲药 “十二时辰” 醇提液皮肤用药的慢性毒性实验研究 [J]. 中国民族民间医药, 2020, 29(1):21−24. ZHOU C Q, ZHAO L, JIANG C, et al. She medicine “twelve hours” alcohol extract skin drug chronic toxicity experimental study [J]. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2020, 29(1): 21−24.(in Chinese)
[6]	刘思延. 畲药十二时辰复方治疗糖尿病足药效机制及外用软膏剂研制 [D]. 福州: 福建中医药大学, 2020. LIU S Y. The Mechanism of Clematis florida Thunb. var. plena D. Don Treatment of Diabetic Foot and the Tevelopment of External Ointment [D]. Fuzhou: Fujian University of Traditional Chinese Medicine, 2020. (in Chinese)
[7]	朱扶蓉, 何舒澜, 林贵灿. 闽产重瓣铁线莲的生药学鉴别研究 [J]. 时珍国医国药, 2014, 25(11):2685−2687. ZHU F R, HE S L, LIN G C. The pharmacognosy identification of Clematis florida Thunb. var. plena D. Don [J]. Lishizhen Medicine and Materia Medica Research, 2014, 25(11): 2685−2687.(in Chinese)
[8]	叶晓霞, 王小敏, 陈善兰, 等. 中药地枫皮及其伪品假地枫皮的DNA条形码鉴定 [J]. 中国实验方剂学杂志, 2019, 25(15):185−190. YE X X, WANG X M, CHEN S L, et al. Identification of Illicium difengpi and its fake i. jiadifengpi using DNA barcoding [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25(15): 185−190.(in Chinese)
[9]	陈璐, 王清蓉, 敖慧, 等. 适合果实类中药的DNA条形码鉴定方法研究 [J]. 辽宁中医杂志, 2018, 45(5):1028−1030. CHEN L, WANG Q R, AO H, et al. General DNA barcode method research for fruits Chinese drugs [J]. Liaoning Journal of Traditional Chinese Medicine, 2018, 45(5): 1028−1030.(in Chinese)
[10]	宫璐, 黄志海, 张靖, 等. 中药络石藤及其习用品的DNA条形码鉴定 [J]. 世界科学技术-中医药现代化, 2016, 18(8):1413−1418. GONG L, HUANG Z H, ZHANG J, et al. Identification of traditional Chinese medicine Trachelospermum jasminoides and its customary supplies using DNA barcoding [J]. World Science and Technology-Modernization of Traditional Chinese Medicine, 2016, 18(8): 1413−1418.(in Chinese)
[11]	郑夏生, 赖智填, 成金乐. DNA条形码在中药破壁饮片物种鉴定的应用 [J]. 中国现代中药, 2016, 18(7):846−850. ZHENG X S, LAI Z T, CHENG J L. Application of DNA barcoding technology in species identification of ultrafine granular powder of herbal medicine [J]. Modern Chinese Medicine, 2016, 18(7): 846−850.(in Chinese)
[12]	陈永兰. 分子标记技术在中药材鉴定中的应用效果观察 [J]. 环球中医药, 2015, 8(S1):84. CHEN Y L. Observation on the application effect of molecular marker technology in the identification of Chinese medicinal materials [J]. Global Traditional Chinese Medicine, 2015, 8(S1): 84.(in Chinese)
[13]	HON C C, CHOW Y C, ZENG F Y, et al. Genetic authentication of ginseng and other traditional Chinese medicine [J]. Acta Pharmacologica Sinica, 2003, 24(9): 841−846.
[14]	陈文超, 苏琬涵, 刘志高, 等. 铁线莲属植物叶绿体微卫星引物开发及其遗传分析 [J]. 浙江农业学报, 2017, 29(12):2023−2031. CHEN W C, SU W H, LIU Z G, et al. Development and genetic analysis of novel chloroplast microsatellite markers of Clematis [J]. Acta Agriculturae Zhejiangensis, 2017, 29(12): 2023−2031.(in Chinese)
[15]	盛璐, 张逢凯, 潘婷, 等. 不同提取方法的铁线莲属叶片总DNA提取效果 [J]. 林业科技开发, 2014, 28(2):16−19. SHENG L, ZHANG F K, PAN T, et al. Effect of different methods on DNA extraction from leaves of Clematis L [J]. China Forestry Science and Technology, 2014, 28(2): 16−19.(in Chinese)
[16]	屈良鹄, 陈月琴. 生物分子分类检索表—原理与方法 [J]. 中山大学学报(自然科学版), 1999, 38(1):1−6. QU L H, CHEN Y Q. Key to molecular TaxonomyPrinciples and methods [J]. Acta Scientiarum Naturalium Universitatis Sunyatseni, 1999, 38(1): 1−6.(in Chinese)
[17]	徐昌艳, 雷丹丹, 曹旭林, 等. 天冬药材基因组DNA提取方法研究 [J]. 安徽农业科学, 2019, 47(8):171−173. XU C Y, LEI D D, CAO X L, et al. Study on extraction method of genomic DNA from ASPARAGI RADIX [J]. Journal of Anhui Agricultural Sciences, 2019, 47(8): 171−173.(in Chinese)
[18]	中国科学院中国植物志编辑委员会. 中国植物志（第58卷）[M]. 北京: 科学出版社, 1979.
[19]	林雪玲, 郑蓉, 何天友, 等. 台湾牛樟总DNA提取方法研究及其PCR验证 [J]. 安徽农学通报, 2019, 25(2):7−9. LIN X L, ZHENG R, HE T Y, et al. Extraction of total DNA and PCR verification for Cinnamomum kanehirae [J]. Anhui Agricultural Science Bulletin, 2019, 25(2): 7−9.(in Chinese)
[20]	何飞宇. 浅析芒果属植物叶绿体DNA提取方法 [J]. 农村经济与科技, 2019, 30(5):44−45. HE F Y. Methods for extracting chloroplast DNA from mango plants [J]. Rural Economy and Science-Technology, 2019, 30(5): 44−45.(in Chinese)
[21]	周仙莉, 田霞, 沈宁东, 等. 藏茴香总DNA提取方法探究 [J]. 分子植物育种, 2019, 17(7):2217−2222. ZHOU X L, TIAN X, SHEN N D, et al. Study on extraction method of total DNA from Carum carvi [J]. Molecular Plant Breeding, 2019, 17(7): 2217−2222.(in Chinese)
[22]	成小威, 苏一钧, 霍恺森, 等. 甘薯叶绿体分离及其DNA提取 [J]. 江苏师范大学学报(自然科学版), 2019, 37(3):22−25. CHENG X W, SU Y J, HUO K S, et al. Chloroplast isolation and cpDNA extraction f Rom sweetpotato [J]. Journal of Jiangsu Normal University (Natural Science Edition), 2019, 37(3): 22−25.(in Chinese)
[23]	余智城, 何雪娇, 林秀香, 等. 柚叶片DNA提取方法比较 [J]. 福建热作科技, 2019, 44(3):26−28. YU Z C, HE X J, LIN X X, et al. Comparison of DNA extraction methods for grapefruit leaves [J]. Fujian Science & Technology of Tropical Crops, 2019, 44(3): 26−28.(in Chinese)