A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis

PENG Jun; CENG Fanyun; LONG Haibo; HUANG Junsheng; GUO Jianrong

doi:10.15886/j.cnki.rdswxb.2012.03.014

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Volume 3 Issue 3

Sep. 2012

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Article Navigation > Journal of Tropical Biology > 2012 > 3(3): 252-257

PENG Jun, CENG Fanyun, LONG Haibo, HUANG Junsheng, GUO Jianrong. A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis[J]. Journal of Tropical Biology, 2012, 3(3): 252-257. doi: 10.15886/j.cnki.rdswxb.2012.03.014

Citation:

PENG Jun, CENG Fanyun, LONG Haibo, HUANG Junsheng, GUO Jianrong. A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis[J]. Journal of Tropical Biology, 2012, 3(3): 252-257. doi: 10.15886/j.cnki.rdswxb.2012.03.014

A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis

doi: 10.15886/j.cnki.rdswxb.2012.03.014

1. Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences;Ministry of Agriculture Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests;Hainan Key Laboratory of Monitoring and Control of Tropical Agricultural Invasive Alien Pests, Danzhou 571737, China;
2. Department of Plant pathology, China Agricultural University;Ministry of Agriculture Key Laboratory of Plant Pathology, Beijing 100193, China

Received Date: 2012-07-09

Abstract

Tissues of most tropical fruit trees contain polysaccharide and polyphenolics that restrict the extraction of high-quality genomic DNA for further molecular biology analysis. This experiment was conducted to develop a simple, universal and effective modified CTAB method for genomic DNA extraction from tissues of tropical fruit trees for southern blot analysis. This optimized procedure includes two steps. First, before the DNA was extracted, the polysaccharide and polyphenolics were pre-removed through the STE lysis buffer by centrifugation. In addition, possible contamination by secondary metaboliates could be avoided by picking up the cotton-shaped genomic DNA floccules resuspended in liquid at the DNA precipitation step with ethanol. With the modified CTAB method, high-quality DNAs were extracted from young leaves of the three representative tropical fruit trees, banana （Musa spp.）, papaya （Carica papaya L.） and longan （Dimocarpus longan Lour.）. After digested with different restriction enzymes, fragments of banana ethylene receptor gene （GenBank accession No. AF113748）, papaya phytoene desaturase gene （DQ779922） and longan flowering-related LEAFY homologous gene （DQ160214） were used as probes for further Southern blot analysis, respectively. The hybridization bands were clear with high background. The results indicate that the modified CTAB method, with pre-treated conjugation by DNA pick-up, is a simple and effective protocol for genomic DNA isolation from tropical fruit trees, which produces high-quality genomic DNA available for further Southern blot analysis and other tests in molecular biology.
- tropical fruits tree,
- DNA extraction,
- modified CTAB method,
- Southern blot analysis

References

[1]	CRUZ M, WHITKUS R, MOTA-BRAVO L. Tropical tree DNA isolation and amplification[J]. Molecular Ecology, 1995,4:787-789.
[2]	SHARMA A D, GILL P K, SINGH P. DNA isolation from dry and fresh samples of polysaccharide-rich plants[J]. Plant Molecular Biology Reporter, 2002,20(4):415.
[3]	JOBES D V, HURLEY D L, THIEN L B. Plant DNA isolation:a method to efficiently remove polyphenolics, polysaccharides, and RNA[J]. Taxon,1995,44:379-386.
[4]	ANGELES J G C, LAURENA A C, MAETECSON-MENDOZA E. Extraction of genomic DNA from the lipid, polysaccharide, and polyphenol-rich coconut (Cocos nucifera L.)[J]. Plant Molecular Biology Reporter,2005,23:297-298.
[5]	KEB-LLANES M, GONZALEZ G, CHI-MANZANERO B, et al. A rapid and simple method for small-scale DNA extraction in Agavaceae and other tropical plants[J]. Plant Molecular Biology Reporter,2002,20(3):299.
[6]	CONDIT R, HUBBELL S P. Abundance and DNA sequence of two-base repeat regions in tropical tree genomes[J]. Genome, 1991,34(1):66-71.
[7]	WOODHEAD M, DAVIES H V, BRENNAN R M, et al. The isolation of genomic DNA from black currant (Ribes nigrum L.)[J]. Molecular Biotechnology,1998,9:243-246.
[8]	CHENG Y J, GUO W W, YI H L, et al. An efficient protocol for genomic DNA extraction from citrus species[J]. Plant Molecular Biology Reporter,2003,21:177-178.
[9]	AHMED I, ISLAM M, ARSHAD W, et al. High-quality plant DNA extraction for PCR:an easy approach[J]. Journal of Applied Genetics,2009,50:105-107.
[10]	KOTCHONI S O, GACHOMO E W. A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants[J]. Plant Molecular Biology Reporter,2009,36:1633-1636.
[11]	冯斗, 张春发, 张颖. 香蕉乙烯受体基因cDNA的克隆及其表达分析[J]. 热带作物学报, 2004(1):6-10.
[12]	YAN P, GAO X Z, SHEN W T, et al. Cloning and expression analysis of phytoene desaturase and ζ-carotene desaturase genes in Carica papaya[J]. Molecular Biology Reports, 2011, 38:785-791.
[13]	曾黎辉, 王庆莲, 洪自同, 等. 龙眼LEAFY同源基因片段的克隆和表达[J]. 热带作物学报, 2006(4):69-73.
[14]	CHENG F S, BROWN S K, WEEDEN N F. A DNA extraction protocol from various tissues in woody species[J]. HortScience, 1997, 32:921-922.
[15]	CHUNG C H, KWON O C, YI Y B, et al. Isolation of quality genomic DNA from tenacious seeds of sesame and perilla[J]. Plant Tissue Culture and Biotechnology, 1998, 4:42-48.
[16]	GAWEL N J, JARRET R L. A modified CTAB DNA extraction procedure for Musa and Ipomoea[J]. Plant Molecular Biology Reporter, 1991, 3:262-266.
[17]	POREBSKI S, GRANT BAILEY L, BAUM B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J]. Plant Molecular Biology Reporter, 1997, 15:8-15.
[18]	DEL SAL G, MANFIOLETTI G, SCHNEIDER C. The CTAB-DNA precipitation method:a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing[J]. Biotechniques, 1989, 7(5):514-520.
[19]	陈桂信, 潘东明, 吕柳新, 等. 果树核DNA提取、目的基因分离与克隆技术研究进展[J]. 福建农林大学学报:自然科学版, 2002, 31(1):44-50.
[20]	赵琳娜, 胡凤月, 吴孝槐, 等. 用于转基因检测的番木瓜基因组DNA提取方法的比较[J]. 现代食品科技, 2010, 26(2):188-191.
[21]	易干军, 于晓英, 霍合强, 等. 香蕉种质资源的AFLP鉴别与分类中DNA模板的制备[J]. 果树学报, 2001, 18(6):345-348.
[22]	肖璇, 孙敏, 王燕, 等. 顽拗植物龙眼基因组DNA提取方法的研究[J]. 生物技术, 2005, 15(1):44-47.
[23]	MACE E S, BUHARIWALLA H K, CROUCH J H. A high-throughput DNA extraction protocol for tropical molecular breeding programs[J]. Plant Molecular Biology Reporter, 2003, 21:459-460.

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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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A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis

1. Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences;Ministry of Agriculture Key Laboratory of Monitoring and Control of Tropical Agricultural and Forest Invasive Alien Pests;Hainan Key Laboratory of Monitoring and Control of Tropical Agricultural Invasive Alien Pests, Danzhou 571737, China;

2. Department of Plant pathology, China Agricultural University;Ministry of Agriculture Key Laboratory of Plant Pathology, Beijing 100193, China

Received Date: 2012-07-09

Key words:

Abstract: Tissues of most tropical fruit trees contain polysaccharide and polyphenolics that restrict the extraction of high-quality genomic DNA for further molecular biology analysis. This experiment was conducted to develop a simple, universal and effective modified CTAB method for genomic DNA extraction from tissues of tropical fruit trees for southern blot analysis. This optimized procedure includes two steps. First, before the DNA was extracted, the polysaccharide and polyphenolics were pre-removed through the STE lysis buffer by centrifugation. In addition, possible contamination by secondary metaboliates could be avoided by picking up the cotton-shaped genomic DNA floccules resuspended in liquid at the DNA precipitation step with ethanol. With the modified CTAB method, high-quality DNAs were extracted from young leaves of the three representative tropical fruit trees, banana （Musa spp.）, papaya （Carica papaya L.） and longan （Dimocarpus longan Lour.）. After digested with different restriction enzymes, fragments of banana ethylene receptor gene （GenBank accession No. AF113748）, papaya phytoene desaturase gene （DQ779922） and longan flowering-related LEAFY homologous gene （DQ160214） were used as probes for further Southern blot analysis, respectively. The hybridization bands were clear with high background. The results indicate that the modified CTAB method, with pre-treated conjugation by DNA pick-up, is a simple and effective protocol for genomic DNA isolation from tropical fruit trees, which produces high-quality genomic DNA available for further Southern blot analysis and other tests in molecular biology.

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Reference (23)

[1]	CRUZ M, WHITKUS R, MOTA-BRAVO L. Tropical tree DNA isolation and amplification[J]. Molecular Ecology, 1995,4:787-789.
[2]	SHARMA A D, GILL P K, SINGH P. DNA isolation from dry and fresh samples of polysaccharide-rich plants[J]. Plant Molecular Biology Reporter, 2002,20(4):415.
[3]	JOBES D V, HURLEY D L, THIEN L B. Plant DNA isolation:a method to efficiently remove polyphenolics, polysaccharides, and RNA[J]. Taxon,1995,44:379-386.
[4]	ANGELES J G C, LAURENA A C, MAETECSON-MENDOZA E. Extraction of genomic DNA from the lipid, polysaccharide, and polyphenol-rich coconut (Cocos nucifera L.)[J]. Plant Molecular Biology Reporter,2005,23:297-298.
[5]	KEB-LLANES M, GONZALEZ G, CHI-MANZANERO B, et al. A rapid and simple method for small-scale DNA extraction in Agavaceae and other tropical plants[J]. Plant Molecular Biology Reporter,2002,20(3):299.
[6]	CONDIT R, HUBBELL S P. Abundance and DNA sequence of two-base repeat regions in tropical tree genomes[J]. Genome, 1991,34(1):66-71.
[7]	WOODHEAD M, DAVIES H V, BRENNAN R M, et al. The isolation of genomic DNA from black currant (Ribes nigrum L.)[J]. Molecular Biotechnology,1998,9:243-246.
[8]	CHENG Y J, GUO W W, YI H L, et al. An efficient protocol for genomic DNA extraction from citrus species[J]. Plant Molecular Biology Reporter,2003,21:177-178.
[9]	AHMED I, ISLAM M, ARSHAD W, et al. High-quality plant DNA extraction for PCR:an easy approach[J]. Journal of Applied Genetics,2009,50:105-107.
[10]	KOTCHONI S O, GACHOMO E W. A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants[J]. Plant Molecular Biology Reporter,2009,36:1633-1636.
[11]	冯斗, 张春发, 张颖. 香蕉乙烯受体基因cDNA的克隆及其表达分析[J]. 热带作物学报, 2004(1):6-10.
[12]	YAN P, GAO X Z, SHEN W T, et al. Cloning and expression analysis of phytoene desaturase and ζ-carotene desaturase genes in Carica papaya[J]. Molecular Biology Reports, 2011, 38:785-791.
[13]	曾黎辉, 王庆莲, 洪自同, 等. 龙眼LEAFY同源基因片段的克隆和表达[J]. 热带作物学报, 2006(4):69-73.
[14]	CHENG F S, BROWN S K, WEEDEN N F. A DNA extraction protocol from various tissues in woody species[J]. HortScience, 1997, 32:921-922.
[15]	CHUNG C H, KWON O C, YI Y B, et al. Isolation of quality genomic DNA from tenacious seeds of sesame and perilla[J]. Plant Tissue Culture and Biotechnology, 1998, 4:42-48.
[16]	GAWEL N J, JARRET R L. A modified CTAB DNA extraction procedure for Musa and Ipomoea[J]. Plant Molecular Biology Reporter, 1991, 3:262-266.
[17]	POREBSKI S, GRANT BAILEY L, BAUM B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J]. Plant Molecular Biology Reporter, 1997, 15:8-15.
[18]	DEL SAL G, MANFIOLETTI G, SCHNEIDER C. The CTAB-DNA precipitation method:a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing[J]. Biotechniques, 1989, 7(5):514-520.
[19]	陈桂信, 潘东明, 吕柳新, 等. 果树核DNA提取、目的基因分离与克隆技术研究进展[J]. 福建农林大学学报:自然科学版, 2002, 31(1):44-50.
[20]	赵琳娜, 胡凤月, 吴孝槐, 等. 用于转基因检测的番木瓜基因组DNA提取方法的比较[J]. 现代食品科技, 2010, 26(2):188-191.
[21]	易干军, 于晓英, 霍合强, 等. 香蕉种质资源的AFLP鉴别与分类中DNA模板的制备[J]. 果树学报, 2001, 18(6):345-348.
[22]	肖璇, 孙敏, 王燕, 等. 顽拗植物龙眼基因组DNA提取方法的研究[J]. 生物技术, 2005, 15(1):44-47.
[23]	MACE E S, BUHARIWALLA H K, CROUCH J H. A high-throughput DNA extraction protocol for tropical molecular breeding programs[J]. Plant Molecular Biology Reporter, 2003, 21:459-460.

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A Modified DNA Extraction Method for Tropical Fruit Trees and Southern Blot Analysis

doi: 10.15886/j.cnki.rdswxb.2012.03.014

Abstract

References

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通讯作者: 陈斌, bchen63@163.com

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