Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

TIAN Sheng-ping; WANG Yang-dong; CHEN Yi-cun; ZHAN Zhi-yong; SI Lin-lin

Volume 25 Issue 2

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Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China
2.
School of Life Science, Anhui Agricultural University, Hefei 230036, Anhui, China

Received Date: 2011-07-13

Abstract

The effect of DNA extraction was analyzed by comparing the young leaf, terminal bud and flower of Litsea cubeba. The time of DNA digestion and several key factors affecting the PCR selective amplification such as Mg2+ concentration, dNTPs concentration and the mount of the selective amplification primer were also trialed. An optimized AFLP reaction system of Litsea cubeba was established. The results showed that the high quality genomic DNA as a PCR template could be isolated from the bud tissue; genomic DNA could be digested in a hour by 5 U EcoR I and 5 U Mse I; The optical selection amplification system was 20 μL reaction mix containing 1.0 U rTaq polymerase, 2.0 μL 10×PCR buffer, 1.8 μL 25 mmol·L-1MgCl2, 1.4 μL 2.5 mmol·L-1dNTP, 100 ng·μL-1 primer each 1.0 μL. Clear and stable amplification band patterns can be obtained and 10 pairs of AFLP primers with good genetic diversity were selected according to the optimized reaction system. The results will be an effective protocol for further studying the genetic structure and differentiation of Litsea cubeba population.
- Litsea cubeba,
- genomic DNA,
- AFLP,
- the polymorphic primer

References

[1]	Mao A A, Wetten A, Fay M, et al. In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree[J].Plant Cell Report, 2000, 19(3):263-267
[2]	王旭, 杨关锋. 我国山苍子开发利用中存在的问题研究与发展对策[J].经济林研究, 2010, 28(3):136-139
[3]	陈卫军, 龚洵胜, 游小敏. 山苍子播种繁殖及扦插育苗初探[J].经济林研究, 2004, 22(4):59-60
[4]	汤青云, 钟桐生, 汤建国, 等. 山苍子中油脂的提取与利用[J].湘南学院学报, 2004, 25(5):61-63
[5]	Luo M, Jiang L K, Zou G L. Acute and genetic toxicity of essential oil extracted from Litsea cubeba (Lour.) Pers.[J]. Food Prot, 2005, 68(3): 581-584
[6]	黄光文, 卢向阳. 我国山苍子油研究概况[J].湖南科技学院学报, 2005, 26(11): 97-100
[7]	陈铁壁, 袁先友, 张敏, 等. 超临界CO₂ 萃取山苍子核仁油的工艺研究[J].生物质化学工程, 2009, 43(1):5-8
[8]	潘晓杰, 陈卫军, 侯红波. 山苍子资源利用加工现状及开发前景的研究[J].经济林研究, 2003, 21(1):79-80
[9]	Yu Y, Jia Z J, Luo B, et al. The fungicidal terpenoids and essential oil from Litsea cubeba in Tibet[J].Molecules, 2010,15(10):7075-7082
[10]	Rumi K, Pathak M G, Kanjila P B. Physico-chemical characteristics of seed oils of some Litsea species found in north-east India[J]. Natural Product Radiance, 2007, 6(4):297-300
[11]	王发松, 杨得坡, 任三香, 等. 山苍子叶挥发油的化学成分与抗真菌活性[J].中药材, 1999,22(8):400-402
[12]	孙雁霞, 石大兴, 王米力, 等. 山苍子的离体培养和植株再生[J].植物生理学通讯, 2002, 38(4):353
[13]	陈卫军, 龚洵胜, 游小敏, 等. 山苍子播种繁殖及扦插育苗初探[J].经济林研究, 2004, 22(4):59-60
[14]	赵佐敏. 影响山苍子离体快繁效果的主要因素研究[J].安徽农业科学, 2005, 33(9):1650-1652
[15]	Wang Y, Jiang Z T, Li R. Complexation and molecular microcapsules of Litsea cubeba essential oil withβ-cyclodextrin and its derivatives[J]. Eur Food Res Technol, 2009, 228:865-873
[16]	Luo M, Jiang L K, Xiao M, et al. Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques[J]. Acta Biochim Biophys Sin, 2004, 36(4):277-283
[17]	殷志勇, 王秋娟, 贾莹. 山苍子水提物柠檬醛抗哮喘作用的实验研究[J].中国临床药理学与治疗学,2005,11(2):197-201
[18]	肖勇, 李良. 木姜子属挥发油成分及其生物活性研究概况[J].云南化工,2007,34(5):85-92
[19]	蔡海清, 钟世安, 艾海东. 山苍籽核仁油合成生物柴油研究[J].中南大学学报: 自然科学版,2009,40(6):1516-1521
[20]	赵敏, 冯颖, 和锐, 等. 喙尾琵甲DNA提取方法比较与AFLP反应体系建立[J].林业科学研究,2010,23(2):234-240
[21]	Ci X Q, Chen J Q, Li Q M. AFLP and ISSR analysis reveals high genetic variation and inter-population differentiation in fragmented populations of the endangered Litsea szemaois (Lauraceae) from Southwest China[J]. Plant Syst Evol, 2008,273(3-4): 237-246
[22]	Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue[J]. Phytochem Bull, 1987,19:11-15
[23]	王亚军, 吴立山, 厉盛华, 等. 几种药品对聚丙烯酰胺凝胶电泳银染的影响[J].水利渔业, 2005,25(2):13-14
[24]	Vroh Bi I, Harvenght L, Chandelier A, et al. Improved RAPD amplification of recalcitrant plant DNA by the use of activated charcoal during DNA extraction[J].Plant Breed, 1996, 115(3): 205-206
[25]	萨姆布鲁克 J, 弗里奇 E F, 曼尼阿蒂斯 T.分子克隆实验指南[M].金冬雁,黎孟枫,译. 2版.北京,科学出版社,1996
[26]	王斌, 翁曼丽. AFLP的原理及其应用[J].杂交水稻,1996,(5):27-30
[27]	李鹏, 汪阳东, 陈益存, 等. 油桐ISSR-PCR最佳反应体系的建立[J].林业科学研究,2008,21(2):194-199
[28]	萨姆布鲁克 J, 拉赛尔 D W. 分子克隆实验指南[M].黄培堂,王嘉玺,朱厚础,等,译.3版.北京:科学出版社,2002

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China

2. School of Life Science, Anhui Agricultural University, Hefei 230036, Anhui, China

Received Date: 2011-07-13

Available Online: 2012-04-13

Abstract: The effect of DNA extraction was analyzed by comparing the young leaf, terminal bud and flower of Litsea cubeba. The time of DNA digestion and several key factors affecting the PCR selective amplification such as Mg2+ concentration, dNTPs concentration and the mount of the selective amplification primer were also trialed. An optimized AFLP reaction system of Litsea cubeba was established. The results showed that the high quality genomic DNA as a PCR template could be isolated from the bud tissue; genomic DNA could be digested in a hour by 5 U EcoR I and 5 U Mse I; The optical selection amplification system was 20 μL reaction mix containing 1.0 U rTaq polymerase, 2.0 μL 10×PCR buffer, 1.8 μL 25 mmol·L-1MgCl2, 1.4 μL 2.5 mmol·L-1dNTP, 100 ng·μL-1 primer each 1.0 μL. Clear and stable amplification band patterns can be obtained and 10 pairs of AFLP primers with good genetic diversity were selected according to the optimized reaction system. The results will be an effective protocol for further studying the genetic structure and differentiation of Litsea cubeba population.

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Reference (28)

[1]	Mao A A, Wetten A, Fay M, et al. In vitro propagation of Litsea cubeba (Lours.) Pers., a multipurpose tree[J].Plant Cell Report, 2000, 19(3):263-267
[2]	王旭, 杨关锋. 我国山苍子开发利用中存在的问题研究与发展对策[J].经济林研究, 2010, 28(3):136-139
[3]	陈卫军, 龚洵胜, 游小敏. 山苍子播种繁殖及扦插育苗初探[J].经济林研究, 2004, 22(4):59-60
[4]	汤青云, 钟桐生, 汤建国, 等. 山苍子中油脂的提取与利用[J].湘南学院学报, 2004, 25(5):61-63
[5]	Luo M, Jiang L K, Zou G L. Acute and genetic toxicity of essential oil extracted from Litsea cubeba (Lour.) Pers.[J]. Food Prot, 2005, 68(3): 581-584
[6]	黄光文, 卢向阳. 我国山苍子油研究概况[J].湖南科技学院学报, 2005, 26(11): 97-100
[7]	陈铁壁, 袁先友, 张敏, 等. 超临界CO₂ 萃取山苍子核仁油的工艺研究[J].生物质化学工程, 2009, 43(1):5-8
[8]	潘晓杰, 陈卫军, 侯红波. 山苍子资源利用加工现状及开发前景的研究[J].经济林研究, 2003, 21(1):79-80
[9]	Yu Y, Jia Z J, Luo B, et al. The fungicidal terpenoids and essential oil from Litsea cubeba in Tibet[J].Molecules, 2010,15(10):7075-7082
[10]	Rumi K, Pathak M G, Kanjila P B. Physico-chemical characteristics of seed oils of some Litsea species found in north-east India[J]. Natural Product Radiance, 2007, 6(4):297-300
[11]	王发松, 杨得坡, 任三香, 等. 山苍子叶挥发油的化学成分与抗真菌活性[J].中药材, 1999,22(8):400-402
[12]	孙雁霞, 石大兴, 王米力, 等. 山苍子的离体培养和植株再生[J].植物生理学通讯, 2002, 38(4):353
[13]	陈卫军, 龚洵胜, 游小敏, 等. 山苍子播种繁殖及扦插育苗初探[J].经济林研究, 2004, 22(4):59-60
[14]	赵佐敏. 影响山苍子离体快繁效果的主要因素研究[J].安徽农业科学, 2005, 33(9):1650-1652
[15]	Wang Y, Jiang Z T, Li R. Complexation and molecular microcapsules of Litsea cubeba essential oil withβ-cyclodextrin and its derivatives[J]. Eur Food Res Technol, 2009, 228:865-873
[16]	Luo M, Jiang L K, Xiao M, et al. Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques[J]. Acta Biochim Biophys Sin, 2004, 36(4):277-283
[17]	殷志勇, 王秋娟, 贾莹. 山苍子水提物柠檬醛抗哮喘作用的实验研究[J].中国临床药理学与治疗学,2005,11(2):197-201
[18]	肖勇, 李良. 木姜子属挥发油成分及其生物活性研究概况[J].云南化工,2007,34(5):85-92
[19]	蔡海清, 钟世安, 艾海东. 山苍籽核仁油合成生物柴油研究[J].中南大学学报: 自然科学版,2009,40(6):1516-1521
[20]	赵敏, 冯颖, 和锐, 等. 喙尾琵甲DNA提取方法比较与AFLP反应体系建立[J].林业科学研究,2010,23(2):234-240
[21]	Ci X Q, Chen J Q, Li Q M. AFLP and ISSR analysis reveals high genetic variation and inter-population differentiation in fragmented populations of the endangered Litsea szemaois (Lauraceae) from Southwest China[J]. Plant Syst Evol, 2008,273(3-4): 237-246
[22]	Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue[J]. Phytochem Bull, 1987,19:11-15
[23]	王亚军, 吴立山, 厉盛华, 等. 几种药品对聚丙烯酰胺凝胶电泳银染的影响[J].水利渔业, 2005,25(2):13-14
[24]	Vroh Bi I, Harvenght L, Chandelier A, et al. Improved RAPD amplification of recalcitrant plant DNA by the use of activated charcoal during DNA extraction[J].Plant Breed, 1996, 115(3): 205-206
[25]	萨姆布鲁克 J, 弗里奇 E F, 曼尼阿蒂斯 T.分子克隆实验指南[M].金冬雁,黎孟枫,译. 2版.北京,科学出版社,1996
[26]	王斌, 翁曼丽. AFLP的原理及其应用[J].杂交水稻,1996,(5):27-30
[27]	李鹏, 汪阳东, 陈益存, 等. 油桐ISSR-PCR最佳反应体系的建立[J].林业科学研究,2008,21(2):194-199
[28]	萨姆布鲁克 J, 拉赛尔 D W. 分子克隆实验指南[M].黄培堂,王嘉玺,朱厚础,等,译.3版.北京:科学出版社,2002

Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

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通讯作者: 陈斌, bchen63@163.com

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Primer Screening and AFLP Amplification Reaction System of Litsea cubeba

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