Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART

doi:10.1016/S1671-2927(11)60087-4

Journal of Integrative Agriculture ›› 2011, Vol. 10 ›› Issue (7): 1004-1009.DOI: 10.1016/S1671-2927(11)60087-4

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

收稿日期:2010-06-08 出版日期:2011-07-21 发布日期:2011-08-14
通讯作者: Correspondence LIU Sheng-yi, Professor, Tel/Fax: +86-27-86812896, E-mail: liusy@oilcrops.cn

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

Received:2010-06-08 Online:2011-07-21 Published:2011-08-14
Contact: Correspondence LIU Sheng-yi, Professor, Tel/Fax: +86-27-86812896, E-mail: liusy@oilcrops.cn

摘要/Abstract

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.

关键词:

text-autospace: " align="left">DSN, full-length library, normalization, oil accumulation, Sesamue indicum, Zhongzhi 14 cDNA library, switching mechanism, SMARTTM

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.

Key words: DSN, full-length library, normalization, oil accumulation, Sesamue indicum, Zhongzhi 14 cDNA library, switching mechanism, SMART_TM

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

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Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART

Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART

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