LIU Xiaoying,LU Changhua,LI Jiahui,et al.Optimization of AFLP Reaction System and Screening of Primers for Pogostemon cablin[J].Northern Horticulture,2020,44(11):117-123.[doi:10.11937/bfyy.20193400]
广藿香AFLP反应体系的优化及引物筛选
- Title:
- Optimization of AFLP Reaction System and Screening of Primers for Pogostemon cablin
- 文献标志码:
- A
- 摘要:
- 以广藿香叶子为试材,采用AFLP分子标记方法,研究了广藿香AFLP反应体系,包括酶切连接体系、连接产物稀释倍数、预扩增产物稀释倍数等关键因素,并对AFLP引物组合(E/M组合)进行筛选,以期为后续的广藿香优良种质资源筛选奠定基础。结果表明:酶切体系为20 μL,含300 ng模板DNA,在37 ℃下反应3 h;连接反应在16 ℃下反应12 h;连接产物的最适稀释倍数为3倍;预扩增产物的最适稀释倍数为20倍。采用优化好的反应体系筛选出了12对条带清晰、多态性丰富的引物组合。优化好的反应体系适用于广藿香AFLP分子标记研究,筛选出的引物也具有较好的多态性,可为后续的广藿香优良种质筛选提供技术支持。
- Abstract:
- The Pogostemon cablin leaves were used as the test material and the AFLP molecular labeling method was used to study the AFLP reaction system of Pogostemon cablin,including key factors such as reaction system of digestion and ligation dilution multiple of ligation product and pre-amplification product,and then screened the AFLP primer combinations (E/M combinations) in order to lay the foundation for the subsequent screening of superior germplasm resources of P.cablin.The results showed that the digestion system was 20 μL,containing 300 ng of template DNA,and reacted at 37 ℃ for 3 hours;the ligation reaction was carried out at 16 ℃ for 12 hours;the optimal dilution multiple of the ligation product was 3 times;the optimal dilution multiple of the pre-amplified product was 20 times.12 pairs of primer combinations with clear and polymorphic bands were screened by optimized reaction system.The optimized reaction system was suitable for the AFLP analysis of P.cablin,and the selected primers also had good polymorphism,which can provide technical support for the subsequent screening of excellent germplasm of P.cablin.
参考文献/References:
[1]国家药典委员会.中华人民共和国药典[M].北京:化学工业出版社,2015:45.[2]徐雯,吴艳清,丁浩然,等.广藿香的药理作用及机制研究进展[J].上海中医药杂志,2017,51(10):103-106.[3]罗兵,孙海燕,徐港明,等.SSR分子标记研究进展[J].安徽农业科学,2013,41(12):5210-5212,5246.[4]VOS P,HOGERS R,BLEEKER M,et al.AFLP:A new technique for DNA fingerprinting[J].Nucl Acids Res,1995,23(21):4407-4414.[5]黄映萍.DNA分子标记研究进展[J].中山大学研究生学刊(自然科学?医学版),2010,31(2):27-36.[6]LI S F,WANG L J,DENG C L,et al.Identification of male-specific AFLP and SCAR markers in the dioecious plant,Humulus scandens[J].Molecular and Cellular Probes,2017,34:68-70.[7]MENG F,PENG M,WANG R,et al.Analysis of genetic diversity in Aconitum kongboense L.revealed by AFLP markers[J].Biochemical Systematics and Ecology,2014,57:388-394.[8]GAUDEUL M,DELAHAYE T,MULLER S.AFLP markers show low levels of clonal propagation and high genotypic diversity in the rare,southern most populations of Linnaea borealis L.(Caprifoliaceae) in the Western Alps[J].Genetica.2019,147:79-90.[9]梁宏伟,王长忠,李忠,等.聚丙烯酰胺凝胶快速、高效银染方法的建立[J].遗传,2008(10):1379-1382.
相似文献/References:
[1]何梦玲,何芳,熊洋,等.茉莉酸甲酯对广藿香叶中百秋里醇含量的影响[J].北方园艺,2014,38(05):147.
HE Meng-ling,HE Fang,XIONG Yang,et al.Effect of Methyl Jasmonate on the Content of Patchouli Alcohol From the Leaves of Pogostemon cablin (Blanco) Benth.[J].Northern Horticulture,2014,38(11):147.
[2]唐堃,李明,赵盼,等.广藿香挥发油对其组织培养幼苗化感自毒作用的研究[J].北方园艺,2014,38(19):128.
TANG Kun,LI Ming,ZHAO Pan,et al.Allelopathy of Effect Volatile Oil from Pogostemon cablin to Its Tissue Culture Seedlings[J].Northern Horticulture,2014,38(11):128.
[3]严寒静,熊洋,张宏意,等.广藿香八倍体叶片叶绿素荧光的日变化[J].北方园艺,2015,39(24):153.[doi:10.11937/bfyy.201524042]
YAN Hanjing,XIONG Yang,ZHANG Hongyi,et al.Diurnal Changes in Chlorophyll Fluorescence Parameters of Homologous Octoploid Pogostemon cablin Leaves[J].Northern Horticulture,2015,39(11):153.[doi:10.11937/bfyy.201524042]
[4]吴燕燕,李明,黄结雯,等.重茬土壤、枯叶腐解液对广藿香扦插苗生理生化指标的影响[J].北方园艺,2017,41(10):149.[doi:10.11937/bfyy.201710034]
WU Yanyan,LI Ming,HUANG Jiewen,et al.Effect of Continuous Cropping Soil and Decomposed Liquid of Dead Leaves on Physiological and Biochemical Indexes of Pogostemon cablin Cutting Seedlings[J].Northern Horticulture,2017,41(11):149.[doi:10.11937/bfyy.201710034]
[5]周界,潘丽萍,李明.广藿香间作紫苏对其连作障碍的缓解效应[J].北方园艺,2020,44(13):111.[doi:10.11937/bfyy.20192582]
ZHOU Jie,PAN Liping,LI Ming.Relieving Effects of Pogostemon cablin Intercropping WithPerilla frutescens on Its Continuous Cropping Obstacle[J].Northern Horticulture,2020,44(11):111.[doi:10.11937/bfyy.20192582]
[6]欧晓华,刘晓莹,李嘉慧,等.两种化学型广藿香叶显微特征比较[J].北方园艺,2021,(03):136.[doi:10.11937/bfyy.20200932]
OU Xiaohua,LIU Xiaoying,LI Jiahui,et al.Comparative on Leaf Microcharacteristics of Two Chemotypes of Pogostemon cablin[J].Northern Horticulture,2021,(11):136.[doi:10.11937/bfyy.20200932]
[7]卢红,李明,李龙明.广藿香植株水提液的化感自毒作用[J].北方园艺,2021,(16):108.[doi:10.11937/bfyy.20210042]
LU Hong,LI Ming,LI Longming.Allelotoxic Effect of Aqueous Extract Solution From Pogostemon cablin[J].Northern Horticulture,2021,(11):108.[doi:10.11937/bfyy.20210042]
备注/Memo
第一作者简介:刘晓莹(1994-),女,硕士研究生,研究方向为药品食品分析与质量控制。E-mail:1552436667@qq.com.责任作者:何梦玲(1975-),女,博士,副教授,硕士生导师,现主要从事中药资源开发与品质评价等研究工作。E-mail:hmldf@126.com.基金项目:国家自然科学基金资助项目(81773829);广东省科技计划资助项目(2015A030302084,2017A030303082,2017A030303081);广东省中医药管理局资助项目(20163010)。收稿日期:2019-11-29