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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.
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Received: 08 June 2010
Accepted:
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Corresponding Authors:
Correspondence LIU Sheng-yi, Professor, Tel/Fax: +86-27-86812896, E-mail: liusy@oilcrops.cn
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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.
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