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Journal of Integrative Agriculture  2011, Vol. 10 Issue (7): 1004-1009    DOI: 10.1016/S1671-2927(11)60087-4
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Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART
Key Laboratory for Oil Crops Biology, Ministry of Agriculture/Oil Crops Research Institute, Chinese Academy of Agricultural Sciences
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摘要  Sesame (Sesamue indicum L.) is one of the most important oilseed crops with high oil yield. Here, we described a simpleand efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14,during its oil accumulation stages. It combined switching mechanism at 5´-end of RNA transcript (SMART) technique andduplex-specific nuclease (DSN) normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processedby normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligationmixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0×106 clonesin this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA libraryconstructed successfully was a full-length library with high quality, and could be used to screen the genes related todevelopment of oil synthesis.

Abstract  Sesame (Sesamue indicum L.) is one of the most important oilseed crops with high oil yield. Here, we described a simpleand efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14,during its oil accumulation stages. It combined switching mechanism at 5´-end of RNA transcript (SMART) technique andduplex-specific nuclease (DSN) normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processedby normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligationmixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0×106 clonesin this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA libraryconstructed successfully was a full-length library with high quality, and could be used to screen the genes related todevelopment of oil synthesis.
Keywords:  DSN      full-length library      normalization      oil accumulation      Sesamue indicum      Zhongzhi 14 cDNA library      switching mechanism      SMARTTM  
Received: 08 June 2010   Accepted:
Corresponding Authors:  Correspondence LIU Sheng-yi, Professor, Tel/Fax: +86-27-86812896, E-mail: liusy@oilcrops.cn   

Cite this article: 

KE Tao; DONG Cai-hua; MAO Han; ZHAO Ying-zhong; LIU Hong-yan and LIU Sheng-yi. 2011. Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART. Journal of Integrative Agriculture, 10(7): 1004-1009.

[1]       Barnes W M. 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophagetemplates. Proceedings of the National Academy of Sciencesof the USA, 91, 2216-2220.
[2]       Cooney R V, Custer L J, Okinaka L, Franke A A. 2001. Effectsof dietary sesame seeds on plasma tocopherol levels. Nutritionand Cancer, 39, 66-71.
[3]       Dorothea B. 2003. Evolution of sesame revisited: domestication,diversity and prospects. Genetic Resources and CropEvolution, 50, 779-787.
[4]       Draper M P, August P R, Connolly T, Packard B, Call K M.2002. Efficient cloning of full-length cDNAs based on cDNAsize fractionation. Genomics, 79, 603-607.
[5]       Gurskaya N G, Diatchenko L, Chenchik A, Siebert P D,Khaspekov G L, Lukyanov K A, Vagner L L, Ermolaeva OD, Lukyanov S A, Sverdlov E D. 1996. Equalizing cDNAsubtraction based on selective suppression of polymerasechain reaction: cloning of Jurkat cell transcripts induced byphytohemaglutinin and phorbol 12-myristate 13-acetate.Analytical Biochemistry, 240, 90-97.
[6]       Harmatha J, Nawrot J. 2002. Insect feeding deterrent activity oflignans and related phenylpropanoids with amethylenedioxyphenyl (piperonyl) structure moiety.Entomologia Experimentalis et Applicata, 104, 56-60.
[7]       Kang M H, Naito M, Sakai K, Uchida K, Osawa T. 2000. Modeof action of sesame lignans in protecting low-densitylipoprotein against oxidative damage in vitro. Life Sciences,66, 161-71.
[8]       Levesque V, Fayad T, Ndiaye K, Nahé Diouf M, Lussier J G.2003. Size-selection of cDNA libraries for the cloning ofcDNAs after suppression subtractive hybridization.Biotechniques, 35, 72-78.
[9]       Matz M, Shagin D, Bogdanova E, Lukyanov S, Diatchenko L,Chenchik A. 1999. Amplification of cDNA ends based ontemplate-switching effect and step-out PCR. Nucleic AcidsResearch, 27, 1558-1560.
[10]    Miyahara Y, Hibasami H, Katsuzaki H, Imai K, Komiya T.2001. Sesamolin from sesame seed inhibits proliferation byinducing apoptosis in human lymphoid leukemia Molt 4Bcells. International Journal of Molecular Medicine, 7, 369-371.
[11]    Ohara O, Temple G. 2001. Directional cDNA library constructionassisted by the in vitro recombination reaction. Nucleic AcidsResearch, 29, e22.Shagin D A, Rebrikov D V, Kozhemyako V B, Altshuler I M,Shcheglov A S, Zhulidov P A, Bogdanova E A, Staroverov DB, Rasskazov V A, Lukyanov S. 2002. A novel method forSNP detection using a new duplex-specific nuclease fromcrab hepatopancreas. Genome Research, 12, 1935-1942.
[12]    Shimizu S, Akimoto K, Shinmen Y, Kawashima H, Sugano M,Yamada H. 1991. Sesamin is a potent and specific inhibitorof Δ5 desaturase in polyunsaturated fatty acid biosynthesis.Lipids, 26, 512-515.
[13]    Shyu Y S, Hwang L S. 2002. Antioxidative activity of the crudeextract of lignan glycosides from unroasted Burma blacksesame meal. Food Research International, 35, 357-365.
[14]    Spiess A N, Ivell R. 2002. A highly efficient method for longchaincDNA synthesis using trehalose and betaine. AnalyticalBiochemistry, 301, 168-174.
[15]    Suh M C, Kim M J, Hur C G, Bae J M, Park Y I, Chung C H,Kang C W, Ohlrogge J B. 2003. Comparative analysis ofexpressed sequence tags from Sesamum indicum andArabidopsis thaliana developing seeds. Plant MolecularBiology, 52, 1107-1123.
[16]    Suzuki Y, Sugano S. 2001. Construction of full-length-enrichedcDNA libraries. The oligo-capping method. Methods inMolecular Biology, 175, 143-153.
[17]    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A,Sugano S. 1997. Construction and characterization of a fulllength-enriched and a 5´-end-enriched cDNA library. Gene,200, 149-156.
[18]    Umezawa T, Sakurai T, Totoki Y, Toyoda A, Seki M, IshiwataA, Akiyama K, Kurotani A, Yoshida T, Mochida K, et al.2008. Sequencing and analysis of approximately 40 000soybean cDNA clones from a full-length-enriched cDNAlibrary. DNA Research, 15, 333-346.
[19]    Wellenreuther R, Schupp I, Poustka A, Wiemann S. 2004. GermancDNA Consortium. SMART amplification combined withcDNA size fractionation in order to obtain large full-lengthclones. BMC Genomics, 5, 36-44.
[20]    Wiemann S, Mehrle A, Bechtel S, Wellenreuther R, PepperkokR, Poustka A. 2003. cDNAs for functional genomics andproteomics: the German Consortium. Comptes RendusBiologies, 326, 1003-1009.
[21]    Xie Y F, Wang B C, Li B, Cai Y F, Xie L, Xia Y X, Chang P A,Jiang H Z. 2007. Construction of cDNA library of cottonmutant Xiangmian-18 library during gland forming stage.Colloids and Surfaces B: Biointerfaces, 60, 258-263.
[22]    Zhao Y Z, Liu H Y. 2007. Breeding of sesame Zhongzhi 14.Crops, 5, 93. (in chinese)Zhu Y Y, Machleder E M, Chenchik A, Li R, Siebert P D. 2001.Reverse transcriptase template switching: a SMARTapproach for full-length cDNA library construction.Biotechniques, 30, 892-897.
[23]   Zhulidov P A, Bogdanova E A, Shcheglov A S, Vagner L L,Khaspekov G L, Kozhemyako V B, Matz M V,Meleshkevitch E, Moroz L L, Lukyanov S A, Shagin D A.2004. Simple cDNA normalization using kamchatka crabduplex-specific nuclease. Nucleic Acids Research, 32, e37.
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