摘要
Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and 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 and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.
Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and 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 and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.
基金
supported by the National Basic Research Program of China (2011cb109305)
the Genetically Modified Organisms Breeding Major Projects, China (2009zx08004-002B)
the Open Project Program of Key Laboratory for Oil Crops Biology, the Ministry of Agriculture, China (200703)
the Foundation of Oil Crops Research Institute, Chinese Academy of Agricultural Sciences
